Continuous production of monoclonal antibody in a packed-bed bioreactor.

PubMed

Golmakany, Naghmeh; Rasaee, Mohammad Javad; Furouzandeh, Mehdi; Shojaosadati, Seyed Abbas; Kashanian, Soheila; Omidfar, Kobra

2005-06-01

In the present study the growth and MAb (monoclonal antibody) production of a mouse x mouse hybridoma cell producing anti-digoxin MAb was evaluated. The hybridoma cells entrapped within the support matrix Fibra-Cel were cultured in batch and continuous mode following special protocols. Cell-culture studies were performed in a 1-litre spinner basket containing 3 g.litre-1 support matrix. Batch culture was operated with the cell density of 42x10(6) cells. During the 7 days of culture, the medium was sampled daily in order to assess glucose and MAb concentrations and the lactate dehydrogenase released into the culture medium. After a culture period of 72 h, the cell density and MAb concentration were found to be 10.4x10(7) cells/3 g of NWPF (non-woven polyester fibre) discs and 250 microg/ml respectively. This yield gradually decreased to 0.55x10(6) cells/3 g of packaging material and 60 microg/ml respectively at the end of the batch culture. In the continuous-culture studies, the batch culture was initially operated for 64.5 h and then continuous flow was started at the dilution rates of 0.15, 0.2, 0.25 and 0.3 day-1 and finally stabilized at 0.25 day-1 within 288 h (12 days). The MAb concentration at steady state was found to be 116-120 microg/day per ml, and the yield of operation was 62.5 mg/day per ml, which was 3.5 times higher than that of batch culture. In conclusion, a packed-bed bioreactor with the support matrix Fibra-Cel, operated in continuous-feeding mode, is more efficient for large-scale MAb production than a batch culture. On the other hand, by using a continuous-culture system, a better supply of nutrients and removal of inhibitory metabolites and proteolytic enzymes was obtained.

Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)

PubMed Central

Dormady, Shane P.; Zhang, Xin-Min; Basch, Ross S.

2000-01-01

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively γ-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be γ-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells. PMID:11050245

A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.

PubMed

Knedlitschek, G; Schneider, F; Gottwald, E; Schaller, T; Eschbach, E; Weibezahn, K F

1999-02-01

Special microenvironmental conditions are required to induce and/or maintain specific qualities of differentiated cells. An important parameter is the three-dimensional tissue architecture that cannot be reproduced in conventional monolayer systems. Advanced tissue culture systems will meet many of these demands, but may reach their limits, especially when gradients of specific substances over distinct tissue layers must be established for long-term culture. These limitations may be overcome by incorporating microstructures into tissue-like culture systems. The microstructured cell support presented consists of a flat array of 625 cubic microcontainers with porous bottoms, in which cells can be supplied with specific media from both sides of the tissue layer. Permanent cell lines and primary rat hepatocytes have been used to test the culture system. In order to define reproducible conditions for tissue formation and for cell adherence to the structure, several ECM (extracellular matrix) components were tested for coating of microstructured substrata. The described tissue culture system offers great flexibility in adapting the cell support to specific needs.

Hair cell recovery in mitotically blocked cultures of the bullfrog saccule

NASA Technical Reports Server (NTRS)

Baird, R. A.; Burton, M. D.; Fashena, D. S.; Naeger, R. A.

2000-01-01

Hair cells in many nonmammalian vertebrates are regenerated by the mitotic division of supporting cell progenitors and the differentiation of the resulting progeny into new hair cells and supporting cells. Recent studies have shown that nonmitotic hair cell recovery after aminoglycoside-induced damage can also occur in the vestibular organs. Using hair cell and supporting cell immunocytochemical markers, we have used confocal and electron microscopy to examine the fate of damaged hair cells and the origin of immature hair cells after gentamicin treatment in mitotically blocked cultures of the bullfrog saccule. Extruding and fragmenting hair cells, which undergo apoptotic cell death, are replaced by scar formations. After losing their bundles, sublethally damaged hair cells remain in the sensory epithelium for prolonged periods, acquiring supporting cell-like morphology and immunoreactivity. These modes of damage appear to be mutually exclusive, implying that sublethally damaged hair cells repair their bundles. Transitional cells, coexpressing hair cell and supporting cell markers, are seen near scar formations created by the expansion of neighboring supporting cells. Most of these cells have morphology and immunoreactivity similar to that of sublethally damaged hair cells. Ultrastructural analysis also reveals that most immature hair cells had autophagic vacuoles, implying that they originated from damaged hair cells rather than supporting cells. Some transitional cells are supporting cells participating in scar formations. Supporting cells also decrease in number during hair cell recovery, supporting the conclusion that some supporting cells undergo phenotypic conversion into hair cells without an intervening mitotic event.

Hair cell recovery in mitotically blocked cultures of the bullfrog saccule

PubMed Central

Baird, Richard A.; Burton, Miriam D.; Fashena, David S.; Naeger, Rebecca A.

2000-01-01

Hair cells in many nonmammalian vertebrates are regenerated by the mitotic division of supporting cell progenitors and the differentiation of the resulting progeny into new hair cells and supporting cells. Recent studies have shown that nonmitotic hair cell recovery after aminoglycoside-induced damage can also occur in the vestibular organs. Using hair cell and supporting cell immunocytochemical markers, we have used confocal and electron microscopy to examine the fate of damaged hair cells and the origin of immature hair cells after gentamicin treatment in mitotically blocked cultures of the bullfrog saccule. Extruding and fragmenting hair cells, which undergo apoptotic cell death, are replaced by scar formations. After losing their bundles, sublethally damaged hair cells remain in the sensory epithelium for prolonged periods, acquiring supporting cell-like morphology and immunoreactivity. These modes of damage appear to be mutually exclusive, implying that sublethally damaged hair cells repair their bundles. Transitional cells, coexpressing hair cell and supporting cell markers, are seen near scar formations created by the expansion of neighboring supporting cells. Most of these cells have morphology and immunoreactivity similar to that of sublethally damaged hair cells. Ultrastructural analysis also reveals that most immature hair cells had autophagic vacuoles, implying that they originated from damaged hair cells rather than supporting cells. Some transitional cells are supporting cells participating in scar formations. Supporting cells also decrease in number during hair cell recovery, supporting the conclusion that some supporting cells undergo phenotypic conversion into hair cells without an intervening mitotic event. PMID:11050201

Hair cell recovery in mitotically blocked cultures of the bullfrog saccule.

PubMed

Baird, R A; Burton, M D; Lysakowski, A; Fashena, D S; Naeger, R A

2000-10-24

Hair cells in many nonmammalian vertebrates are regenerated by the mitotic division of supporting cell progenitors and the differentiation of the resulting progeny into new hair cells and supporting cells. Recent studies have shown that nonmitotic hair cell recovery after aminoglycoside-induced damage can also occur in the vestibular organs. Using hair cell and supporting cell immunocytochemical markers, we have used confocal and electron microscopy to examine the fate of damaged hair cells and the origin of immature hair cells after gentamicin treatment in mitotically blocked cultures of the bullfrog saccule. Extruding and fragmenting hair cells, which undergo apoptotic cell death, are replaced by scar formations. After losing their bundles, sublethally damaged hair cells remain in the sensory epithelium for prolonged periods, acquiring supporting cell-like morphology and immunoreactivity. These modes of damage appear to be mutually exclusive, implying that sublethally damaged hair cells repair their bundles. Transitional cells, coexpressing hair cell and supporting cell markers, are seen near scar formations created by the expansion of neighboring supporting cells. Most of these cells have morphology and immunoreactivity similar to that of sublethally damaged hair cells. Ultrastructural analysis also reveals that most immature hair cells had autophagic vacuoles, implying that they originated from damaged hair cells rather than supporting cells. Some transitional cells are supporting cells participating in scar formations. Supporting cells also decrease in number during hair cell recovery, supporting the conclusion that some supporting cells undergo phenotypic conversion into hair cells without an intervening mitotic event.

Sustained release of stem cell factor in a double network hydrogel for ex vivo culture of cord blood-derived CD34+ cells.

PubMed

Zhang, Yuanhao; Pan, Xiuwei; Shi, Zhen; Cai, Haibo; Gao, Yun; Zhang, Weian

2018-04-01

Stem cell factor (SCF) is considered as a commonly indispensable cytokine for proliferation of haematopoietic stem cells (HSCs), which is used in large dosages during ex vivo culture. The work presented here aimed to reduce the consumption of SCF by sustained release but still support cells proliferation and maintain the multipotency of HSCs. Stem cell factor was physically encapsulated within a hyaluronic acid/gelatin double network (HGDN) hydrogel to achieve a slow release rate. CD34 + cells were cultured within the SCF-loaded HGDN hydrogel for 14 days. The cell number, phenotype and functional capacity were investigated after culture. The HGDN hydrogels had desirable properties and encapsulated SCF kept being released for more than 6 days. SCF remained the native bioactivity, and the proliferation of HSCs within the SCF-loaded HGDN hydrogel was not affected, although the consumption of SCF was only a quarter in comparison with the conventional culture. Moreover, CD34 + cells harvested from the SCF-loaded HGDN hydrogels generated more multipotent colony-forming units (CFU-GEMM). The data suggested that the SCF-loaded HGDN hydrogel could support ex vivo culture of HSCs, thus providing a cost-effective culture protocol for HSCs. © 2017 John Wiley & Sons Ltd.

Microfluidic engineered high cell density three-dimensional neural cultures

NASA Astrophysics Data System (ADS)

Cullen, D. Kacy; Vukasinovic, Jelena; Glezer, Ari; La Placa, Michelle C.

2007-06-01

Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell-cell/cell-matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 µm) 3D neural cultures supported by passive diffusion, cell densities <=5.0 × 103 cells mm-3 were required for survival. In 3D neuronal and neuronal-astrocytic co-cultures with increased cell density (>=104 cells mm-3), continuous medium perfusion at 2.0-11.0 µL min-1 improved viability compared to non-perfused cultures (p < 0.01), which exhibited widespread cell death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00-6.25 µL min-1 (p < 0.05); however, at perfusion rates of 10.0-11.0 µL min-1 survival did not depend on the distance from the perfusion source, and resulted in a preservation of cell density with >90% viability in both neuronal cultures and neuronal-astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell-cell/cell-matrix interactions.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

PubMed

Wang, Mei; Wu, Chunping; Guo, Yu; Cao, Xiaojuan; Zheng, Wenwei; Fan, Guo-Kang

2017-05-01

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

2010-01-01

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

PubMed Central

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

PubMed

Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

2016-01-01

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithelia of the inner ear.

PubMed

Warchol, Mark E

2002-04-01

Sensory hair cells in the inner ears of nonmammalian vertebrates can regenerate after injury. In many species, replacement hair cells are produced by the proliferation of epithelial supporting cells. Thus, the ability of supporting cells to undergo renewed proliferation is a key determinant of regenerative ability. The present study used cultures of isolated inner ear sensory epithelia to identify cellular signals that regulate supporting cell proliferation. Small pieces of sensory epithelia from the chicken utricle were cultured in glass microwells. Under those conditions, cell proliferation was inversely related to local cell density. The signaling molecules N-cadherin, beta-catenin, and focal adhesion kinase were immunolocalized in the cultured epithelial cells, and high levels of phosphotyrosine immunoreactivity were present at cell-cell junctions and focal contacts of proliferating cells. Binding of microbeads coated with a function-blocking antibody to N-cadherin inhibited ongoing proliferation. The growth of epithelial cells was also affected by the density of extracellular matrix molecules. The results suggest that cell density, cell-cell contact, and the composition of the extracellular matrix may be critical influences on the regulation of sensory regeneration in the inner ear.

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

PubMed Central

Chu, Pat P. Y.; Bari, Sudipto; Fan, Xiubo; Gay, Florence P. H.; Ang, Justina M. L.; Chiu, Gigi N. C.; Lim, Sai K.; Hwang, William Y. K.

2012-01-01

Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture. PMID:22775077

Embryonic Stem Cells: Isolation, Characterization and Culture

NASA Astrophysics Data System (ADS)

Amit, Michal; Itskovitz-Eldor, Joseph

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

PubMed Central

Takenaka, Chiemi; Miyajima, Hiroshi; Yoda, Yusuke; Imazato, Hideo; Yamamoto, Takako; Gomi, Shinichi; Ohshima, Yasuhiro; Kagawa, Kenichi; Sasaki, Tetsuji; Kawamata, Shin

2015-01-01

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control. PMID:26115194

Role of Somatic Testicular Cells during Mouse Spermatogenesis in Three-Dimensional Collagen Gel Culture System

PubMed Central

Khajavi, Noushafarin; Akbari, Mohammad; Abolhassani, Farid; Dehpour, Ahmad Reza; Koruji, Morteza; Habibi Roudkenar, Mehryar

2014-01-01

Objective Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. Materials and Methods In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed. Results Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Conclusion Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro. PMID:24518977

Growing B Lymphocytes in a Three-Dimensional Culture System

NASA Technical Reports Server (NTRS)

Wu, J. H. David; Bottaro, Andrea

2010-01-01

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing within 3D cultures that have been modified to foster lymphopoiesis retain an immunophenotype that closely recapitulates cells in fresh bone marrow harvests. The 3D culture system has been found to be capable of supporting long-lived (8 weeks) populations of B and T lymphocytes from peripheral lymphoid organs, in the absence of activation signals, to an extent not achievable by conventional culture techniques. Interestingly, it has been found that 3D-culture B cells display a phenotype that has characteristics of both B1a and B2 cells. These promising preliminary observations suggest that the 3D culture system could be used with success in the study of peripheral-B-lymphocyte biology and in the development of biotechnological techniques and processes.

Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape.

PubMed

Kamperman, Tom; Henke, Sieger; Visser, Claas Willem; Karperien, Marcel; Leijten, Jeroen

2017-06-01

Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off-center encapsulated cells. High-speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off-center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long-term culture of individual cells within <5-µm-thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell-centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biogelx: Cell Culture on Self-Assembling Peptide Gels.

PubMed

Harper, Mhairi M; Connolly, Michael L; Goldie, Laura; Irvine, Eleanore J; Shaw, Joshua E; Jayawarna, Vineetha; Richardson, Stephen M; Dalby, Matthew J; Lightbody, David; Ulijn, Rein V

2018-01-01

Aromatic peptide amphiphiles can form self-supporting nanostructured hydrogels with tunable mechanical properties and chemical compositions. These hydrogels are increasingly applied in two-dimensional (2D) and three-dimensional (3D) cell culture, where there is a rapidly growing need to store, grow, proliferate, and manipulate naturally derived cells within a hydrated, 3D matrix. Biogelx Limited is a biomaterials company, created to commercialize these bio-inspired hydrogels to cell biologists for a range of cell culture applications. This chapter describes methods of various characterization and cell culture techniques specifically optimized for compatibility with Biogelx products.

Algal culture studies related to a Closed Ecological Life Support System (CELSS)

NASA Technical Reports Server (NTRS)

Radmer, R. O.; Ollinger, O.; Venables, A.; Fernandez, E.

1982-01-01

Studies with algal cultures which relate to closed ecological life support systems (CELSS) are discussed. A description of a constant cell density apparatus for continuous culture of algae is included. Excretion of algal by-products, and nitrogen utilization and excretion are discussed.

Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor*

PubMed Central

Liu, Zhong-wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

2014-01-01

This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and activation of cellular signaling pathways in GDNF synthesis and secretion in the culturing systems were also assessed and compared with a conventional culturing system. In this study, we found that the ZrO2 ceramic foam culturing system was biocompatible, using which the GDNF yields were elevated and sustained by stimulated cell proliferation and activation of signaling pathways in astrocytes cultured in the system. In conclusion, the ZrO2 ceramic foam is promising for the development of a GDNF mass production device for Parkinson’s disease treatment. PMID:25471830

Sequential growth factor application in bone marrow stromal cell ligament engineering.

PubMed

Moreau, Jodie E; Chen, Jingsong; Horan, Rebecca L; Kaplan, David L; Altman, Gregory H

2005-01-01

In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

PubMed Central

Mossel, Eric C.; Wang, Jieru; Jeffers, Scott; Edeen, Karen E.; Wang, Shuanglin; Cosgrove, Gregory P.; Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Pearson, Leonard D.; Holmes, Kathryn V.; Mason, Robert J.

2008-01-01

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 hours and peaked at approximately 105 pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection. PMID:18022664

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

PubMed Central

Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

2016-01-01

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

Differentiation-associated alteration in human monocyte-macrophage accessory cell function.

PubMed

Mayernik, D G; Ul-Haq, A; Rinehart, J J

1983-05-01

Human monocyte (Mo) to macrophage (Mx) differentiation is associated with marked and well studied changes in morphology, biochemical parameters, and effector cell function. Nevertheless, the comparative accessory cell (AC) function of blood Mo and differentiated Mx has not been carefully studied. We, therefore, examined the kinetics and mechanisms of change in AC function during in vitro Mo to Mx differentiation. The system utilized has two distinctive features: blood Mo and resultant cultured Mx represent a cohort of cells derived from the bone marrow within a 12-hr period. Moreover, the in vitro derived Mx utilized herein have been characterized extensively and are functionally and biochemically similar to pulmonary macrophages (PMx). In the experiments reported, AC functions of blood Mo, Mx derived from Mo after 1 to 6 days of culture, and PMx was compared. AC were cultured with nylon wool column-purified autologous T cells and were stimulated with concanavalin A (Con A) or streptokinase-streptodornase (SKSD). Blood T cell proliferation to Con A or SKSD was inhibited greater than 90% by the removal of Mo and was reconstituted by 20% Mo. Mx derived from Mo by culture for 1 to 3 days exhibited the same (or better) AC function as Mo when T cells were stimulated with either SKSD or Con A. In marked contrast, Mx derived from 6-day cultures exhibited less than or equal to 15% of Mo (i.e., control) capacity to support T cell proliferative response to SKSD. Six-day Mx support T cell proliferation to Con A was somewhat variable. Similar to 6-day cultured Mx, PMx failed to function as AC. The mechanism of loss of AC function was examined: a) cultured Mx maintained Ia antigen positivity for greater than 8 days; b) mixing experiments with Mo + 6-day cultured Mx or Mo + PMx demonstrated no T cell suppression; c) the normal capacity of most 6-day cultured Mx to support Con A but not SKSD induced T cell proliferation, apparently ruled out the loss of the ability to deliver a nonspecific "second signal" as the involved mechanism; d) inhibition of Mo to Mx differentiation by dexamethasone preserved AC activity. Thus, human culture-derived Mx and PMx exhibit deficit AC function through loss of an undefined mechanism. However, loss of AC antigen processing or presentation may occur.

NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure.

PubMed

Lei, Xiao-hua; Ning, Li-na; Cao, Yu-jing; Liu, Shuang; Zhang, Shou-bing; Qiu, Zhi-fang; Hu, Hui-min; Zhang, Hui-shan; Liu, Shu; Duan, En-kui

2011-01-01

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory" environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

INCIDENCE AND DETECTION OF PLEUROPNEUMONIA-LIKE ORGANISMS IN CELL CULTURES BY FLUORESCENT ANTIBODY AND CULTURAL PROCEDURES1

PubMed Central

Barile, Michael F.; Malizia, Walter F.; Riggs, Donald B.

1962-01-01

Barile, Michael F. (National Institutes of Health, Bethesda, Md.), Walter F. Malizia, and Donald B. Riggs. Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol. 84:130–136. 1962—A total of 102 tissue-cell cultures from 17 separate laboratories was examined for pleuropneumonia-like organisms (PPLO) by the fluorescent antibody and cultural procedures. PPLO were isolated from 48 of the 49 tissue-cell cultures found positive for PPLO by the fluorescent antibody procedure, and results of the two procedures agreed in 101 of the 102 (99%) cases. PPLO were isolated from none of 10 primary-cell cultures prepared from six animal species and from 48 of 92 (52%) continuous-cell cultures prepared from eight animal species. Cells grown in media containing antibiotics were more frequently contaminated with PPLO (72%) than cells grown in antibiotic-free media (7%). Cultures (91%) from tissue-culture-producing laboratories and cultures (76%) used for propagation of microorganisms were contaminated with PPLO, although none used for tissue-culture metabolic studies was contaminated. In addition, our findings support the view that PPLO contamination of cell cultures is probably owing to bacterial contaminants which revert to L forms in the presence of antibiotics. Images PMID:13865001

Elastic hydrogel substrate supports robust expansion of murine myoblasts and enhances their engraftment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Ke, E-mail: dk1118@yeah.net; Yang, Zhong; Xu, Jian-zhong, E-mail: xjzspine@163.com

The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate formore » satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic hydrogels. • Myoblasts expanded on the elastic hydrogel exhibited enhanced in vivo engraftment.« less

Method and Apparatus for a Miniature Bioreactor System for Long-Term Cell Culture

NASA Technical Reports Server (NTRS)

Kleis, Stanley J. (Inventor); Geffert, Sandra K. (Inventor); Gonda, Steve R. (Inventor)

2015-01-01

A bioreactor and method that permits continuous and simultaneous short, moderate, or long term cell culturing of one or more cell types or tissue in a laminar flow configuration is disclosed, where the bioreactor supports at least two laminar flow zones, which are isolated by laminar flow without the need for physical barriers between the zones. The bioreactors of this invention are ideally suited for studying short, moderate and long term studies of cell cultures and the response of cell cultures to one or more stressors such as pharmaceuticals, hypoxia, pathogens, or any other stressor. The bioreactors of this invention are also ideally suited for short, moderate or long term cell culturing with periodic cell harvesting and/or medium processing for secreted cellular components.

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture.

PubMed

Goff, A K; Smith, L C

1998-04-01

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.

Protocol for culturing low density pure rat hippocampal neurons supported by mature mixed neuron cultures.

PubMed

Yang, Qian; Ke, Yini; Luo, Jianhong; Tang, Yang

2017-02-01

primary hippocampal neuron cultures allow for subcellular morphological dissection, easy access to drug treatment and electrophysiology analysis of individual neurons, and is therefore an ideal model for the study of neuron physiology. While neuron and glia mixed cultures are relatively easy to prepare, pure neurons are particular hard to culture at low densities which are suitable for morphology studies. This may be due to a lack of neurotrophic factors such as brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and Glial cell line-derived neurotrophic factor (GDNF). In this study we used a two step protocol in which neuron-glia mixed cultures were initially prepared for maturation to support the growth of young neurons plated at very low densities. Our protocol showed that neurotrophic support resulted in physiologically functional hippocampal neurons with larger cell body, increased neurite length and decreased branching and complexity compared to cultures prepared using a conventional method. Our protocol provides a novel way to culture highly uniformed hippocampal neurons for acquiring high quality, neuron based data. Copyright © 2016 Elsevier B.V. All rights reserved.

A fibrin-supported myocardial organ culture for isolation of cardiac stem cells via the recapitulation of cardiac homeostasis.

PubMed

Kim, Jong-Tae; Chung, Hye Jin; Seo, Ji-Yeon; Yang, Young-Il; Choi, Min-Young; Kim, Hyeong-In; Yang, Tae-Hyun; Lee, Won-Jin; Youn, Young Chul; Kim, Hye Jung; Kim, Yeon Mee; Lee, Hyukjin; Jang, Yang-Soo; Lee, Seung-Jin

2015-04-01

There is great interest in the development of cardiac stem cells (CSCs) cell-based therapeutics; thus, clinical translation requires an efficient method for attaining therapeutic quantities of these cells. Furthermore, an in vitro model to investigate the mechanisms regulating the cardiac homeostasis is crucial. We sought to develop a simple myocardial culture method for enabling both the recapitulation of myocardial homeostasis and the simultaneous isolation of CSCs. The intact myocardial fragments were encapsulated 3-dimensionally into the fibrin and cultured under dynamic conditions. The fibrin provided secure physical support and substratum to the myocardium, which mediated integrin-mediated cell signaling that allowed in situ renewal, outgrowth and cardiomyogenic differentiation of CSCs, mimicking myocardial homeostasis. Since our culture maintained the myocardial CSCs niches, it was possible to define the identity of in vitro renewed CSCs that situated in the interstitium between cardiomyocytes and microvessels. Lastly, the use of matrix-restricted fibrinolysis enabled the selective isolation of outgrown CSCs that retained the clonogenicity, long-term growth competency and cardiovascular commitment potential. Collectively, this myocardial culture might be used as an alternative tool for studying cardiac biology and developing cell-based therapeutics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Canine Adipose-Derived Mesenchymal Stromal/Stem Cells in Serum-Free Medium.

PubMed

Liu, Zhuoming; Screven, Rudell; Boxer, Lynne; Myers, Michael J; Devireddy, Lax R

2018-06-20

In this article, we report on the development of a defined serum-free medium capable of supporting the culture expansion of mesenchymal stromal/stem cells (MSCs) from canine adipose tissue (canine Ad-MSCs). The potential benefits of serum-free media can only be utilized if cells cultured in serum-free media maintain the same functional characteristics as cells cultured in serum-containing media. Therefore, we analyze the characteristics of canine Ad-MSCs cultured in this serum-free medium or in serum-containing medium through evaluation of growth kinetics, clonogenic capacity, senescence, and differentiation capacity. Both, serum-containing medium and our serum-free medium, supported efficient growth and colony formation of canine Ad-MSCs. In addition, canine Ad-MSCs cultured in both media demonstrated similar viability after freeze/thaw, similar cell surface marker expression, and were capable of trilineage differentiation. While canine Ad-MSCs cultured in both media were generally similar, under the conditions of our study, canine Ad-MSCs cultured in serum-free medium demonstrated a shorter lag phase and higher colony-forming capacity, accelerated population doubling, maintained multipotentiality at higher passage numbers, and underwent senescence at higher passage numbers compared to canine Ad-MSCs cultured in conventional serum-containing medium. These results suggest that canine Ad-MSCs cultured in serum-free medium retain the basic characteristics associated with canine Ad-MSCs cultured in serum-containing medium, although some differences in growth kinetics were observed.

High-throughput combinatorial cell co-culture using microfluidics.

PubMed

Tumarkin, Ethan; Tzadu, Lsan; Csaszar, Elizabeth; Seo, Minseok; Zhang, Hong; Lee, Anna; Peerani, Raheem; Purpura, Kelly; Zandstra, Peter W; Kumacheva, Eugenia

2011-06-01

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate. This journal is © The Royal Society of Chemistry 2011

Epimorphin Regulates the Intestinal Stem Cell Niche via Effects on the Stromal Microenvironment.

PubMed

Vishy, Courtney E; Swietlicki, Elzbieta A; Gazit, Vered; Amara, Suneetha; Heslop, Gabriela; Lu, Jianyun; Levin, Marc S; Rubin, Deborah C

2018-04-06

Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin (Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission and small bowel length in vivo. Stem cell derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim -/- and WT mice. Enteroids co-cultured with ISEMFS had increased growth and crypt-like budding compared to enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared to co-cultures with WT ISEMFs. In primary crypt cultures, Epim -/- enteroids had significantly increased surface area and budding compared WTs. However stem cell assays comparing the number of Epim -/- vs WT colony forming units after first passage showed no differences in the absence of ISEMF support. Epim -/- vs. WT ISEMFs had increased Wnt4 expression and addition of Wnt4 to WT co-cultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Bioconvection in Cultures of the Calcifying Unicellular Alga Pleurochrysis Carterae

NASA Technical Reports Server (NTRS)

Montufar-Solis, Dina; Duke, P. Jackie; Marsh, Mary E.

2003-01-01

The unicellular, marine, calcifying alga P leurochiysis carterae--a model to study cell morphogenesis, cell polarity, calcification, gravitaxis, reproduction and development-- has extremely flexible culture requirements. Support studies for a flight experiment addressing cell motility suggested that cell density (cells/ml) affects cell movement in P. carterae cultures through the gradual establishment of bioconvection as the culture grows. To assess the effect of cell density on direction of the movement, without the effects of aging of the culture, swimming behavior was analyzed in aliquots from a series of dilutions obtained from a stock culture. Results showed that at low concentrations cells swim randomly. As the concentration increases, upswimming patterns overtake random swimming. Gradually, up and down movement patterns prevail, representative of bioconvection. This oriented swimming of P. carterae occurs in a wide range of concentrations, adding to the list of flexible requirements, in this case, cell concentration, to be used for spaceflight studies addressing cell motility and bioconvection in a unicellular model of biologically directed mineralization.

Biotechnology

NASA Image and Video Library

2003-05-05

Aboard the International Space Station (ISS), the Tissue Culture Medium (TCM) is the bioreactor vessel in which cell cultures are grown. With its two syringe ports, it is much like a bag used to administer intravenous fluid, except it allows gas exchange needed for life. The TCM contains cell culture medium, and when frozen cells are flown to the ISS, they are thawed and introduced to the TCM through the syringe ports. In the Cellular Biotechnology Operations Support System-Fluid Dynamics Investigation (CBOSS-FDI) experiment, several mixing procedures are being assessed to determine which method achieves the most uniform mixing of growing cells and culture medium.

Kidney cell electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

Immobilisation of a thrombopoietin peptidic mimic by self-assembled monolayers for culture of CD34+ cells.

PubMed

Lee, Eun-Ju; Be, Cheang Ly; Vinson, Andrew R; Riches, Andrew G; Fehr, Friederike; Gardiner, James; Gengenbach, Thomas R; Winkler, David A; Haylock, David

2015-01-01

Compared to soluble cytokines, surface-tethered ligands can deliver biological signalling with precise control of spatial positioning and concentration. A strategy that immobilises ligand molecules on a surface in a uniform orientation using non-cleavable linkages under physiological conditions would enhance the specific and systemic delivery of signalling in the local environment. We used mixed self-assembled monolayers (SAMs) of oxyamine- and oligo(ethylene glycol)-terminated thiols on gold to covalently install aldehyde- or ketone-functionalised ligands via oxime conjugation. Characterisation by electrochemistry and X-ray photoelectron spectroscopy showed quantitative immobilisation of the ligands on SAM surfaces. The thrombopoietin mimetic peptide, RILL, was immobilised on SAMs and the bioactivity of the substrate was demonstrated by culturing factor-dependent cells. We also optimised the immobilisation and wash conditions so that the peptide was not released into the culture medium and the immobilised RILL could be re-used for consecutive cell cultures. The surface also supported the growth of haematopoietic CD34+ cells comparable to the standard thrombopoietin-supplemented culture. Furthermore, the RILL-immobilised SAM surface was as effective in expanding uncommitted CD34+ cells as standard culture. The stimulatory effect of surface-tethered ligands in haematopoietic stem cell expansion supports the use of ligand immobilisation strategies to replicate the haematopoietic stem cell niche. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture.

PubMed

Perestrelo, Ana Rubina; Grenha, Ana; Rosa da Costa, Ana M; Belo, José António

2014-07-01

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro differentiation assay revealed that mouse ESCs cultured in LBG preserve their tri-lineage differentiation capacity. In conclusion, our data indicate that LBG coating promotes mouse ESC growth in an undifferentiated state demonstrating to be a viable, non-animal derived alternative to gelatin to support pluripotent mouse ESCs in culture. Copyright © 2014 Elsevier B.V. All rights reserved.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

PubMed

Futrega, Kathryn; Atkinson, Kerry; Lott, William B; Doran, Michael R

2017-04-01

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34 + progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34 + cell expansion outcomes. By contrast, a substantial increase in CD34 + CD38 - cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34 + CD38 - cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34 + CD38 - cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34 + cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

Multi-Cellular 3D Human Primary Liver Cell Cultures Elevate Metabolic Activity Under Fluidic Flow

PubMed Central

Esch, Mandy B.; Prot, Jean-Matthieu; Wang, Ying I.; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A.; Applegate, Dawn R.

2015-01-01

Predicting drug-induced liver injury with in vitro cell culture models more accurately would be of significant value to the pharmaceutical industry. To this end we have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop and bidirectional fluid flow (average flow rate of 650 μL/min, and a maximum shear stress of 0.64 dyne/cm2). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures derived from human tissues increase their metabolic activity in response to bidirectional fluidic flow. Since bidirectional flow drastically changes the behavior of other cells types that are shear sensitive, the finding that bidirectional flow increases the metabolic activity of primary liver cells also supports the theory that this increase in metabolic activity is likely caused by increased levels of gas and metabolite exchange or by the accumulation of soluble growth factors rather than by shear sensing. Our results indicate that device operation with bi-directional gravity-driven medium flow supports the 14-day culture of a mix of primary human liver cells with the benefits of enhanced metabolic activity. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs. PMID:25857666

Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors.

PubMed

Khurshid, Madiha; Mulet-Sierra, Aillette; Adesida, Adetola; Sen, Arindom

2018-03-01

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair. Copyright © 2017 John Wiley & Sons, Ltd.

From Three-Dimensional Cell Culture to Organs-on-Chips

PubMed Central

Huh, Dongeun; Hamilton, Geraldine A.; Ingber, Donald E.

2014-01-01

Three-dimensional (3D) cell culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional two-dimensional (2D) culture systems. Here, we review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These ‘organs-on-chips’ permit study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing. PMID:22033488

Culture Models for Studying Thyroid Biology and Disorders

PubMed Central

Toda, Shuji; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Matsunobu, Aki; Yamamoto, Mihoko; Ootani, Akifumi; Yamasaki, Fumio; Koike, Eisuke; Sugihara, Hajime

2011-01-01

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types. PMID:22363871

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold.

PubMed

Hassanein, Wessam; Uluer, Mehmet C; Langford, John; Woodall, Jhade D; Cimeno, Arielle; Dhru, Urmil; Werdesheim, Avraham; Harrison, Joshua; Rivera-Pratt, Carlos; Klepfer, Stephen; Khalifeh, Ali; Buckingham, Bryan; Brazio, Philip S; Parsell, Dawn; Klassen, Charlie; Drachenberg, Cinthia; Barth, Rolf N; LaMattina, John C

2017-01-02

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

Gravity, chromosomes, and organized development in aseptically cultured plant cells

NASA Technical Reports Server (NTRS)

Krikorian, Abraham D.

1993-01-01

The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

Akins, Robert E.; Schroedl, Nancy A.; Gonda, Steve R.; Hartzell, Charles R.

1994-01-01

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

1997-01-01

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

The infection of primary avian tracheal epithelial cells with infectious bronchitis virus

PubMed Central

Shen, Ching-I; Wang, Ching-Ho; Liao, Jiunn-Wang; Hsu, Tien-Wang; Kuo, Shu-Ming; Su, Hong-Lin

2009-01-01

Here we introduce a culture system for the isolation, passaging and amplification of avian tracheal epithelial (ATE) cells. The ATE medium, which contains chicken embryo extract and fetal bovine serum, supports the growth of ciliated cells, goblet cells and basal cells from chicken tracheas on fibronectin- or matrigel-coated dishes. Non-epithelial cells make up less than 10% of the total population. We further show that ATE cells support the replication and spread of infectious bronchitis virus (IBV). Interestingly, immunocytostaining revealed that basal cells are resistant to IBV infection. We also demonstrate that glycosaminoglycan had no effect on infection of the cells by IBV. Taken together, these findings suggest that primary ATE cells provide a novel cell culture system for the amplification of IBV and the in vitro characterization of viral cytopathogenesis. PMID:19793537

Tunable swelling of polyelectrolyte multilayers in cell culture media for modulating NIH-3T3 cells adhesion.

PubMed

Qi, Wei; Cai, Peng; Yuan, Wenjing; Wang, Hua

2014-11-01

For polyelectrolyte multilayers (PEMs) assembled by the layer-by-layer (LbL) assembly technique, their nanostructure and properties can be governed by many parameters during the building process. Here, it was demonstrated that the swelling of the PEMs containing poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) in cell culture media could be tuned with changing supporting salt solutions during the assembly process. Importantly, the influence of the PEMs assembled in different salt solutions on NIH-3T3 cell adhesion was observable. Specifically, the cells could possess a higher affinity for the films assembled in low salt concentration (i.e. 0.15M NaCl) or no salt, the poorly swelling films in cell culture media, which was manifested by the large cell spreading area and focal adhesions. In contrast, those were assembled in higher salt concentration, highly swelling films in cell culture media, were less attractive for the fibroblasts. As a result, the cell adhesion behaviors may be manipulated by tailoring the physicochemical properties of the films, which could be performed by changing the assembly conditions such as supporting salt concentration. Such a finding might promise a great potential in designing desired biomaterials for tissue engineering and regenerative medicine. © 2014 Wiley Periodicals, Inc.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

PubMed Central

Muraglia, Anita; Nguyen, Van Thi; Nardini, Marta; Mogni, Massimo; Coviello, Domenico; Dozin, Beatrice; Strada, Paolo; Baldelli, Ilaria; Formica, Matteo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-01-01

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype. PMID:29209609

Induced Retro-Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA.

PubMed

Nazemroaya, Fatemeh; Soheili, Zahra-Soheila; Samiei, Shahram; Deezagi, Abdolkhalegh; Ahmadieh, Hamid; Davari, Malihe; Heidari, Razeih; Bagheri, Abouzar; Darvishalipour-Astaneh, Shamila

2017-10-01

Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Co-Culture of Human Endothelial Cells and Foreskin Fibroblasts on 3D Silk-Fibrin Scaffolds Supports Vascularization.

PubMed

Samal, Juhi; Weinandy, Stefan; Weinandy, Agnieszka; Helmedag, Marius; Rongen, Lisanne; Hermanns-Sachweh, Benita; Kundu, Subhas C; Jockenhoevel, Stefan

2015-10-01

A successful strategy to enhance the in vivo survival of engineered tissues would be to prevascularize them. In this study, fabricated silk fibroin scaffolds from mulberry and non-mulberry silkworms are investigated and compared for supporting the co-culture of human umbilical vein endothelial cells and human foreskin fibroblasts. Scaffolds are cytocompatible and when combined with fibrin gel support capillary-like structure formation. Density and interconnectivity of the formed structures are found to be better in mulberry scaffolds. ELISA shows that levels of vascular endothelial growth factor (VEGF) released in co-cultures with fibrin gel are significantly higher than in co-cultures without fibrin gel. RT PCR shows an increase in VEGFR2 expression in mulberry scaffolds indicating these scaffolds combined with fibrin provide a suitable microenvironment for the development of capillary-like structures. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine Sertoli cells.

PubMed

Menegazzo, Massimo; Zuccarello, Daniela; Luca, Giovanni; Ferlin, Alberto; Calvitti, Mario; Mancuso, Francesca; Calafiore, Riccardo; Foresta, Carlo

2011-10-01

Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. Fresh sperm cells (5 ml of 20 × 10(6)/ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Specimen Sample Preservation for Cell and Tissue Cultures

NASA Technical Reports Server (NTRS)

Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

1996-01-01

The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

PubMed

Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

2015-08-01

Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds

PubMed Central

Balakrishnan, Sreenath; Suma, M.S.; Raju, Shilpa R.; Bhargav, Santosh D.B.; Arunima, S.; Das, Saumitra

2015-01-01

Abstract We present a perfusion culture system with miniature bioreactors and peristaltic pumps. The bioreactors are designed for perfusion, live-cell imaging studies, easy incorporation of microfabricated scaffolds, and convenience of operation in standard cell culture techniques. By combining with miniature peristaltic pumps—one for each bioreactor to avoid cross-contamination and to maintain desired flow rate in each—we have made a culture system that facilitates perfusion culture inside standard incubators. This scalable system can support multiple parallel perfusion experiments. The major components are fabricated by three-dimensional printing using VeroWhite, which we show to be amenable to ex vivo cell culture. Furthermore, the components of the system can be reused, thus making it economical. We validate the system and illustrate its versatility by culturing primary rat hepatocytes, live imaging the growth of mouse fibroblasts (NIH 3T3) on microfabricated ring-scaffolds inserted into the bioreactor, performing perfusion culture of breast cancer cells (MCF7), and high-magnification imaging of hepatocarcinoma cells (HuH7). PMID:26309810

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

PubMed Central

Shi, Xiao-Lei; Gu, Jin-Yang; Zhang, Yue; Han, Bing; Xiao, Jiang-Qiang; Yuan, Xian-Wen; Zhang, Ning; Ding, Yi-Tao

2011-01-01

AIM: To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs) could be maintained in serum from acute-on-chronic liver failure (ACLF) patients. METHODS: Hepatocyte supportive functions and cytotoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evaluated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemokine profile was also examined for the normal serum and liver failure serum. RESULTS: Hepatocyte growth factor (HGF) and Tumor necrosis factor; tumor necrosis factor (TNF)-α were remarkably elevated in response to ACLF while epidermal growth factor (EGF) and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver support functions in the homo-hepatocyte culture. Hepatocytes co-cultured with MSCs could tolerate the cytotoxicity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cultured with healthy human serum in vitro. In addition, co-cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum. CONCLUSION: ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro. PMID:21633639

Nanotechnology versus stem cell engineering: in vitro comparison of neurite inductive potentials.

PubMed

Morano, Michela; Wrobel, Sandra; Fregnan, Federica; Ziv-Polat, Ofra; Shahar, Abraham; Ratzka, Andreas; Grothe, Claudia; Geuna, Stefano; Haastert-Talini, Kirsten

2014-01-01

Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation. Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system. We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12 cell system. Our results evidence that nanotechnology as used in our setup is superior over stem cell engineering when it comes to in vitro models for PNR. Furthermore, np-NTFs can easily be suspended in regenerative hydrogel matrix and could be delivered that way to nerve conduits for future in vivo studies and medical application.

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

PubMed Central

Dargaville, Tim R; Hollier, Brett G; Shokoohmand, Ali; Hoogenboom, Richard

2014-01-01

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG). PMID:24714592

Gallium phosphide nanowires as a substrate for cultured neurons.

PubMed

Hällström, Waldemar; Mårtensson, Thomas; Prinz, Christelle; Gustavsson, Per; Montelius, Lars; Samuelson, Lars; Kanje, Martin

2007-10-01

Dissociated sensory neurons were cultured on epitaxial gallium phosphide (GaP) nanowires grown vertically from a gallium phosphide surface. Substrates covered by 2.5 microm long, 50 nm wide nanowires supported cell adhesion and axonal outgrowth. Cell survival was better on nanowire substrates than on planar control substrates. The cells interacted closely with the nanostructures, and cells penetrated by hundreds of wires were observed as well as wire bending due to forces exerted by the cells.

Three Dimensional Neuronal Cell Cultures More Accurately Model Voltage Gated Calcium Channel Functionality in Freshly Dissected Nerve Tissue

PubMed Central

Kisaalita, William

2012-01-01

It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells’ functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns. PMID:23049767

A novel flow-perfusion bioreactor supports 3D dynamic cell culture.

PubMed

Sailon, Alexander M; Allori, Alexander C; Davidson, Edward H; Reformat, Derek D; Allen, Robert J; Warren, Stephen M

2009-01-01

Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm). A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm) scaffolds. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. By day 8, static scaffolds had a periphery cell density of 67% +/- 5.0%, while in the core it was 0.3% +/- 0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94% +/- 8.3% and core density of 76% +/- 3.1% at day 8. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

Integrated strain array for cellular mechanobiology studies

NASA Astrophysics Data System (ADS)

Simmons, C. S.; Sim, J. Y.; Baechtold, P.; Gonzalez, A.; Chung, C.; Borghi, N.; Pruitt, B. L.

2011-05-01

We have developed an integrated strain array for cell culture enabling high-throughput mechano-transduction studies. Biocompatible cell culture chambers were integrated with an acrylic pneumatic compartment and microprocessor-based control system. Each element of the array consists of a deformable membrane supported by a cylindrical pillar within a well. For user-prescribed waveforms, the annular region of the deformable membrane is pulled into the well around the pillar under vacuum, causing the pillar-supported region with cultured cells to be stretched biaxially. The optically clear device and pillar-based mechanism of operation enables imaging on standard laboratory microscopes. Straightforward fabrication utilizes off-the-shelf components, soft lithography techniques in polydimethylsiloxane and laser ablation of acrylic sheets. Proof of compatibility with basic biological assays and standard imaging equipment were accomplished by straining C2C12 skeletal myoblasts on the device for 6 h. At higher strains, cells and actin stress fibers realign with a circumferential preference.

Evaluation of RPE65, CRALBP, VEGF, CD68, and tyrosinase gene expression in human retinal pigment epithelial cells cultured on amniotic membrane.

PubMed

Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Khalooghi, Keynoush; Ahmadieh, Hamid; Kanavi, Mojgan Rezaie; Samiei, Shahram; Pakravesh, Jalil

2011-06-01

The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.

3D culture of murine neural stem cells on decellularized mouse brain sections.

PubMed

De Waele, Jorrit; Reekmans, Kristien; Daans, Jasmijn; Goossens, Herman; Berneman, Zwi; Ponsaerts, Peter

2015-02-01

Transplantation of neural stem cells (NSC) in diseased or injured brain tissue is widely studied as a potential treatment for various neurological pathologies. However, effective cell replacement therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers after transplantation. In this context, it is hypothesized that structural support for grafted NSC will be of utmost importance. With this study, we present a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular three-dimensional (3D) brain sections. Next, the obtained 3D brain sections were seeded with murine NSC expressing both the eGFP and luciferase reporter proteins (NSC-eGFP/Luc). Using real-time bioluminescence imaging, the survival and growth of seeded NSC-eGFP/Luc cells was longitudinally monitored for 1-7 weeks in culture, indicating the ability of the acellular brain sections to support sustained ex vivo growth of NSC. Next, the organization of a 3D maze-like cellular structure was examined using confocal microscopy. Moreover, under mitogenic stimuli (EGF and hFGF-2), most cells in this 3D culture retained their NSC phenotype. Concluding, we here present a novel protocol for decellularization of mouse brain sections, which subsequently support long-term 3D culture of undifferentiated NSC. Copyright © 2014 Elsevier Ltd. All rights reserved.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion

PubMed Central

Futrega, Kathryn; Atkinson, Kerry; Lott, William B.

2017-01-01

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications. PMID:28406754

Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*

PubMed Central

Tape, Christopher J.; Norrie, Ida C.; Worboys, Jonathan D.; Lim, Lindsay; Lauffenburger, Douglas A.; Jørgensen, Claus

2014-01-01

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-KDEL) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDCM.tub-KDEL and LyrM37-KDEL facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell–cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

Supportive development of functional tissues for biomedical research using the MINUSHEET® perfusion system

PubMed Central

2012-01-01

Functional tissues generated under in vitro conditions are urgently needed in biomedical research. However, the engineering of tissues is rather difficult, since their development is influenced by numerous parameters. In consequence, a versatile culture system was developed to respond the unmet needs. Optimal adhesion for cells in this system is reached by the selection of individual biomaterials. To protect cells during handling and culture, the biomaterial is mounted onto a MINUSHEET® tissue carrier. While adherence of cells takes place in the static environment of a 24 well culture plate, generation of tissues is accomplished in one of several available perfusion culture containers. In the basic version a continuous flow of always fresh culture medium is provided to the developing tissue. In a gradient perfusion culture container epithelia are exposed to different fluids at the luminal and basal sides. Another special container with a transparent lid and base enables microscopic visualization of ongoing tissue development. A further container exhibits a flexible silicone lid to apply force onto the developing tissue thereby mimicking mechanical load that is required for developing connective and muscular tissue. Finally, stem/progenitor cells are kept at the interface of an artificial polyester interstitium within a perfusion culture container offering for example an optimal environment for the spatial development of renal tubules. The system presented here was evaluated by various research groups. As a result a variety of publications including most interesting applications were published. In the present paper these data were reviewed and analyzed. All of the results point out that the cell biological profile of engineered tissues can be strongly improved, when the introduced perfusion culture technique is applied in combination with specific biomaterials supporting primary adhesion of cells. PMID:23369669

Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation

NASA Astrophysics Data System (ADS)

Correia, Clara R.; Pirraco, Rogério P.; Cerqueira, Mariana T.; Marques, Alexandra P.; Reis, Rui L.; Mano, João F.

2016-02-01

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals.

PubMed

Sahare, Mahesh G; Suyatno; Imai, Hiroshi

2018-04-01

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans. Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized. In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species-specific requirements for growth factors and mechanisms supporting the self-renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential. Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.

Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

PubMed

Chen, Qiushui; He, Ziyi; Liu, Wu; Lin, Xuexia; Wu, Jing; Li, Haifang; Lin, Jin-Ming

2015-10-28

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

PubMed Central

Tricoli, Lucas; Berry, Deborah L.; Albanese, Chris

2018-01-01

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive “living biobanks” of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research. PMID:28287583

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

PubMed

Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C

2013-01-01

Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Producing biodiesel from cotton seed oil using Rhizopus oryzae ATTC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

USDA-ARS?s Scientific Manuscript database

The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and i...

Constructing kidney-like tissues from cells based on programs for organ development: toward a method of in vitro tissue engineering of the kidney.

PubMed

Rosines, Eran; Johkura, Kohei; Zhang, Xing; Schmidt, Heidi J; Decambre, Marvalyn; Bush, Kevin T; Nigam, Sanjay K

2010-08-01

The plausibility of constructing vascularized three-dimensional (3D) kidney tissue from cells was investigated. The kidney develops from mutual inductive interactions between cells of the ureteric bud (UB), derived from the Wolffian duct (WD), and the metanephric mesenchyme (MM). We found that isolated MMs were capable of inducing branching morphogenesis of the WD (an epithelial tube) in recombination cultures; suggesting that the isolated MM retains inductive capacity for WD-derived epithelial tubule cells other than those from the UB. Hanging drop aggregates of embryonic and adult renal epithelial cells from UB and mouse inner medullary collecting duct cell (IMCD) lines, which are ultimately of WD origin, were capable of inducing MM epithelialization and tubulogenesis with apparent connections (UB cells) and collecting duct-like tubules with lumens (IMCD). This supports the view that the collecting system can be constructed from certain epithelial cells (those ultimately of WD origin) when stimulated by MM. Although the functions of the MM could not be replaced by cultured mesenchymal cells, primary MM cells and one MM-derived cell line (BSN) produced factors that stimulate UB branching morphogenesis, whereas another, rat inducible metanephric mesenchyme (RIMM-18), supported WD budding as a feeder layer. This indicates that some MM functions can be recapitulated by cells. Although engineering of a kidney-like tissue from cultured cells alone remains to be achieved, these results suggest the feasibility of such an approach following the normal developmental progression of the UB and MM. Consistent with this notion, implants of kidney-like tissues constructed in vitro from recombinations of the UB and MM survived for over 5 weeks and achieved an apparently host-derived glomerular vasculature. Lastly, we addressed the issue of optimal macro- and micro-patterning of kidney-like tissue, which might be necessary for function of an organ assembled using a tissue engineering approach. To identify suitable conditions, 3D reconstructions of HoxB7-green fluorescent protein mouse rudiments (E12) cultured on a filter or suspended in a collagen gel (type I or type IV) revealed that type IV collagen 3D culture supports the deepest tissue growth (600 +/- 8 microm) and the largest kidney volume (0.22 +/- 0.02 mm(3)), and enabled the development of an umbrella-shaped collecting system such as occurs in vivo. Taken together with prior work (Rosines et al., 2007; Steer et al., 2002), these results support the plausibility of a developmental strategy for constructing and propagating vascularized 3D kidney-like tissues from recombinations of cultured renal progenitor cells and/or primordial tissue.

Novel "breath figure"-based synthetic PLGA matrices for in vitro modeling of mammary morphogenesis and assessing chemotherapeutic response.

PubMed

Ponnusamy, Thiruselvam; Chakravarty, Geetika; Mondal, Debasis; John, Vijay T

2014-05-01

Biodegradable poly(lactic-co-glycolic acid) (PLGA) porous films are developed to support mammary cell growth and function. Such porous polymer matrices of PLGA are generated using the easily implemented water-templating "breath-figure" technique that allows water droplets to penetrate the nascent polymer films to create a rough porous polymer film. Such breath figure-based micropatterned porous films show higher epithelial differentiation and growth than the corresponding flat 2D films, and represent the first instance of using them for tissue culture. Specifically, the breath figure morphology supports robust acinar growth with almost double the number of lobular-alveolar units compared to the 2D cultures. Gene profile analysis indicates that the cells grown on porous polymer films show enhanced expressions of mammary differentiation genes (GATA3, EMA, and INTEGB4) but lower the expression of mesenchymal gene (CALLA). Hormonal stimulation of these cultures dramatically increases expression of progenitor marker gene Notch1. Importantly, cells grown on porous PLGA films exhibit an enhanced resistance to doxorubicin treatment in comparison to 2D cultures. Breath-figure PLGA films show promise in mimicking in vivo mammary functions and can potentially be used to screen chemotherapeutic drugs. The simplicity and ease of fabrication of these polymer films is especially appealing to the development of effective biomaterials to support cell culture and differentiation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jozaki, K.; Kuriu, A.; Hirota, S.

1991-03-01

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3)more » and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.« less

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

PubMed

Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

2016-02-01

To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Bioreactors to Influence Stem Cell Fate: Augmentation of Mesenchymal Stem Cell Signaling Pathways via Dynamic Culture Systems

PubMed Central

Yeatts, Andrew B.; Choquette, Daniel T.; Fisher, John P.

2012-01-01

Background Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro. Scope of Review This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems. Major Conclusions The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased. General Significance Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. PMID:22705676

Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system.

PubMed

Minuth, Will W; Denk, Lucia

2016-03-01

To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET(®) tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written.

Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.

PubMed

Thangaraj, Gopenath; Greif, Alexander; Layer, Paul G

2011-10-01

Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2-3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

PubMed

Trojani, Christophe; Weiss, Pierre; Michiels, Jean-François; Vinatier, Claire; Guicheux, Jérôme; Daculsi, Guy; Gaudray, Patrick; Carle, Georges F; Rochet, Nathalie

2005-09-01

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

The expression of cancer stem cell markers in human colorectal carcinoma cells in a microenvironment dependent manner.

PubMed

Stankevicius, Vaidotas; Kunigenas, Linas; Stankunas, Edvinas; Kuodyte, Karolina; Strainiene, Egle; Cicenas, Jonas; Samalavicius, Narimantas E; Suziedelis, Kestutis

2017-03-18

Numerous lines of evidence support the hierarchical model of cancer development and tumor initiation. According to the theory, cancer stem cells play a crucial role in the formation of the tumor and should be targeted for more effective anticancer treatment. However, cancer stem cells quickly loose their characteristics when propagated as 2D cell culture, indicating that the 2D cell culture does not provide the appropriate settings to maintain an in vivo environment. In this study we have investigated the expression of self-renewal, cancer stem cell and epithelial to mesenchymal transition markers after the transfer of human colorectal carcinoma cell DLD1 and HT29 lines from 2D cell cultures to scaffold-attached laminin rich extracellular matrix and scaffold-free multicellular spheroid 3D culture models. Based on the up-regulated expression of multipotency, CSC and EMT markers, our data suggests that human colorectal carcinoma cells grown in 3D exhibit enhanced cancer stem cell characteristics. Therefore, in order to design more efficient targeted therapies, we suggest that 3D cell culture models should be employed in cancer stem cell research. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

PubMed

Shadforth, Audra M A; Suzuki, Shuko; Theodoropoulos, Christina; Richardson, Neil A; Chirila, Traian V; Harkin, Damien G

2017-06-01

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell ® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na + /K + -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Short-term exposure of umbilical cord blood CD34+ cells to granulocyte-macrophage colony-stimulating factor early in culture improves ex vivo expansion of neutrophils.

PubMed

Marturana, Flavia; Timmins, Nicholas E; Nielsen, Lars K

2011-03-01

Despite the availability of modern antibiotics/antimycotics and cytokine support, neutropenic infection accounts for the majority of chemotherapy-associated deaths. While transfusion support with donor neutrophils is possible, cost and complicated logistics make such an option unrealistic on a routine basis. A manufactured neutrophil product could enable routine prophylactic administration of neutrophils, preventing the onset of neutropenia and substantially reducing the risk of infection. We examined the use of pre-culture strategies and various cytokine/modulator combinations to improve neutrophil expansion from umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HPC). Enriched UCB HPC were cultured using either two-phase pre-culture strategies or a single phase using various cytokine/modulator combinations. Outcome was assessed with respect to numerical expansion, cell morphology, granulation and respiratory burst activity. Pre-culture in the absence of strong differentiation signals (e.g. granulocyte colony-stimulating factor; G-CSF) failed to provide any improvement to final neutrophil yields. Similarly, removal of differentiating cells during pre-culture failed to improve neutrophil yields to an appreciable extent. Of the cytokine/modulator combinations, the addition of granulocyte-macrophage (GM)-colony-stimulating factor (CSF) alone gave the greatest increase. In order to avoid production of monocytes, it was necessary to remove GM-CSF on day 5. Using this strategy, neutrophil expansion improved 2.7-fold. Although all cytokines and culture strategies employed have been reported previously to enhance HPC expansion, we found that the addition of GM-CSF alone was sufficient to improve total cell yields maximally. The need to remove GM-CSF on day 5 to avoid monocyte differentiation highlights the context and time-dependent complexity of exogenous signaling in hematopoietic cell differentiation and growth.

Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies

PubMed Central

Pedersen, Jenny M.; Shim, Yoo-Sik; Hans, Vaibhav; Phillips, Martin B.; Macdonald, Jeffrey M.; Walker, Glenn; Andersen, Melvin E.; Clewell, Harvey J.; Yoon, Miyoung

2016-01-01

Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices – RealBio® and QuasiVivo® (QV) – and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental conditions for long-term hepatocyte cell culture using the QV bioreactor. This system facilitated the use of low system volumes coupled with higher flow rates. This design supports cellular respiration by increasing oxygen concentrations in the vicinity of the cells and facilitates long-term kinetic studies of low clearance test compounds. These two goals were achieved while simultaneously keeping the shear stress experienced by the cells within acceptable limits. PMID:27747210

Creatine supports propagation and promotes neuronal differentiation of inner ear progenitor cells.

PubMed

Di Santo, Stefano; Mina, Amir; Ducray, Angélique; Widmer, Hans R; Senn, Pascal

2014-05-07

Long-term propagation of inner ear-derived progenitor/stem cells beyond the third generation and differentiation into inner ear cell types has been shown to be feasible, but challenging. We investigated whether the known neuroprotective guanidine compound creatine (Cr) promotes propagation of inner ear progenitor/stem cells as mitogen-expanded neurosphere cultures judged from the formation of spheres over passages. In addition, we studied whether Cr alone or in combination with brain-derived neurotrophic factor (BDNF) promotes neuronal differentiation of inner ear progenitors. For this purpose, early postnatal rat spiral ganglia, utricle, and organ of Corti-derived progenitors were grown as floating spheres in the absence (controls) or presence of Cr (5 mM) from passage 3 onward. Similarly, dissociated sphere-derived cultures were differentiated for 14 days in the presence or absence of Cr (5 mM) and spiral ganglia sphere-derived cultures in a combination of Cr with the neurotrophin BDNF (50 ng/ml). We found that the cumulative total number of spheres over all passages was significantly higher after Cr supplementation as compared with controls in all the three inner ear cultures. In contrast, sphere sizes were not affected by the administration of Cr. Administration of Cr during differentiation of spiral ganglia cells resulted in a significantly higher density of β-III-tubulin-positive cells compared with controls, whereas densities of myosin VIIa-positive cells in cultures of utricle and organ of Corti were not affected by the treatment. Importantly, a combination of Cr with the neurotrophin BDNF resulted in further significantly increased densities of β-III-tubulin-positive cells in cultures of spiral ganglia cells as compared with single treatments. In sum, Cr promoted continuing propagation of rat inner ear-derived progenitor cells and supported specifically in combination with BDNF the differentiation of neuronal cell types from spiral ganglion-derived spheres.

Pre-treatment of synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue

PubMed Central

Radisic, Milica; Park, Hyoungshin; Martens, Timothy P.; Salazar-Lazaro, Johanna E.; Geng, Wenliang; Wang, Yadong; Langer, Robert; Freed, Lisa E.; Vunjak-Novakovic, Gordana

2009-01-01

Native myocardium consists of several cell types, of which approximately one-third are myocytes and most of the nonmyocytes are fibroblasts. By analogy with monolayer culture in which fibroblasts were removed to prevent overgrowth, early attempts to engineer myocardium utilized cell populations enriched for cardiac myocytes (CMs; ~80–90% of total cells). We hypothesized that the pre-treatment of synthetic elastomeric scaffolds with cardiac fibroblasts (CFs) will enhance the functional assembly of the engineered cardiac constructs by creating an environment supportive of cardiomyocyte attachment and function. Cells isolated from neonatal rat ventricles were prepared to form three distinct populations: rapidly plating cells identified as CFs, slowly plating cells identified as CMs, and unseparated initial population of cells (US). The cell fractions (3 × 106 cells total) were seeded into poly(glycerol sebacate) scaffolds (highly porous discs, 5 mm in diameter × 2-mm thick) using Matrigel™, either separately (CM or CF), concurrently (US), or sequentially (CF pre-treatment followed by CM culture, CF + CM), and cultured in spinner flasks. The CF + CM group had the highest amplitude of contraction and the lowest excitation threshold, superior DNA content, and higher glucose consumption rate. The CF + CM group exhibited compact 100- to 200-μm thick layers of elongated myocytes aligned in parallel over layers of collagen-producing fibroblasts, while US and CM groups exhibited scattered and poorly elongated myocytes. The sequential co-culture of CF and CM on a synthetic elastomer scaffold thus created an environment supportive of cardiomyocyte attachment, differentiation, and contractile function, presumably due to scaffold conditioning by cultured fibroblasts. When implanted over the infarcted myocardium in a nude rat model, cell-free poly(glycerol sebacate) remained at the ventricular wall after 2 weeks of in vivo, and was vascularized. PMID:18041719

Impaired Endothelial Progenitor Cell Mobilization and Dysfunctional Bone Marrow Stroma in Diabetes Mellitus

PubMed Central

Rafii, Shahin; Jaspers, Janneke E.; White, Ian A.; Hooper, Andrea T.; Doevendans, Pieter A.; Verhaar, Marianne C.

2013-01-01

Background Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired –at least partly– due to dysfunction of the bone marrow stromal compartment. Methods Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1+Flk-1+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34+ hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell–endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. Results In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. Conclusion EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients. PMID:23555959

Huperzine A protects neural stem cells against Aβ-induced apoptosis in a neural stem cells and microglia co-culture system

PubMed Central

Zhu, Ning; Lin, Jizong; Wang, Kewan; Wei, Meidan; Chen, Qingzhuang; Wang, Yong

2015-01-01

Objectives: This study aims to explore whether Huperzine A (HupA) could protect neural stem cells against amyloid beta-peptide Aβ induced apoptosis in a neural stem cells (NSCs) and microglia co-culture system. Methods: Rat NSCs and microglial cells were isolated, cultured and identified with immunofluorescence Assays (IFA). Co-culture systems of NSCs and microglial cells were employed using Transwell Permeable Supports. The effects of Aβ1-42 on NSCs were studied in 4 groups using co-culture systems: NSCs, Aβ+NSCs, co-culture and Aβ+co-culture groups. Bromodeoxyuridine (BrdU) incorporation and flow cytometry were utilized to assess the differences of proliferation, differentiation and apoptosis of NSCs between the groups. LQ test was performed to assess the amounts of IL-6, TNF-α and MIP-α secreted, and flow cytometry and Western blotting were used to assess apoptosis of NSCs and the expressions of Bcl-2 and Bax in each group. Results: IFA results showed that isolated rat NSCs were nestin-positive and microglial cells were CD11b/c-positive. Among all the groups, the Aβ+co-culture group has the lowest BrdU expression level, the lowest MAP2-positive, ChAT-positive cell counts and the highest NSC apoptosis rate. Smaller amounts of IL-6, TNF-α and MIP-α were being secreted by microglial cells in the HupA+Aβ+co-culture group compared with those in the Aβ+ co-culture group. Also the Bcl-2: Bax ratio was much higher in the HupA+Aβ+co-culture group than in the Aβ+co-culture group. Conclusions: HupA inhibits cell apoptosis through restraining microglia’s inflammatory response induced by Aβ1-42. PMID:26261518

Generation and Characterization of JCV Permissive Hybrid Cell Lines

PubMed Central

Sariyer, Ilker K.; Safak, Mahmut; Gordon, Jennifer; Khalili, Kamel

2009-01-01

JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents. PMID:19442856

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

PubMed

Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

2015-07-15

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

Clonal Growth of Dermal Papilla Cells in Hydrogels Reveals Intrinsic Differences between Sox2-Positive and -Negative Cells In Vitro and In Vivo

PubMed Central

Driskell, Ryan R; Juneja, Vikram R; Connelly, John T; Kretzschmar, Kai; Tan, David W -M; Watt, Fiona M

2012-01-01

In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2− DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2−, and CD133−Sox2− (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133− cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2− spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2− cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function. PMID:22189784

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia.

PubMed

Usuludin, Suaidah Binte Mohamed; Cao, Xue; Lim, Mayasari

2012-05-01

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion. Copyright © 2011 Wiley Periodicals, Inc.

Perimysial fibroblasts of extraocular muscle, as unique as the muscle fibers.

PubMed

Kusner, Linda L; Young, Andrew; Tjoe, Steven; Leahy, Patrick; Kaminski, Henry J

2010-01-01

Extraocular muscle (EOM) has a distinct skeletal muscle phenotype. The hypothesis for the study was that fibroblasts support the unique EOM phenotype and that perimysial fibroblasts derived from EOM have properties that distinguish them from fibroblasts derived from other skeletal muscle. Perimysial fibroblasts from leg muscle (LM-Fibro) and EOM (EOM-Fibro) of mice were derived and maintained in culture. EOM- and LM-Fibro were assessed morphologically and for vimentin, smooth muscle actin, and Thy-1 immunoreactivity. DNA microarray analysis was performed on LM- and EOM-Fibro grown in conditions that support myoblast differentiation. To assess trophic interactions, co-cultures of myoblasts from established cell lines, CL-EOM and CL-LM with, EOM- or LM-Fibro were performed in direct contact and in a permeable filter support culture. The degree of myotube maturation was assessed by the percentage of myotubes with more than three myonuclei per myotube. EOM- and LM-Fibro cells exhibited distinct morphologies. Both cell types proliferated as a monolayer and expressed vimentin. Fifty-five percent (SD 4.4%) of EOM-Fibro were Thy-1 positive compared with only 24% (SD 4.4%) of LM-Fibro. DNA microarray analysis demonstrated differential expression of structural, immune response, and metabolism-related genes between EOM- and LM-Fibro. Co-cultures demonstrated that mature myotube formation in EOM-derived cell lines was supported to a greater extent by EOM-Fibro than by LM-Fibro, compared with CL-EOM grown with LM-Fibro. Fibroblasts from EOM demonstrate distinct properties that distinguish them from leg muscle-derived fibroblasts. The distinct properties of EOM-Fibro may support the unique EOM phenotype and contribute to their differential involvement in disease.

Sustainable production of azadirachtin from differentiated in vitro cell lines of neem (Azadirachta indica)

PubMed Central

Singh, Mithilesh; Chaturvedi, Rakhi

2013-01-01

Azadirachtin has high industrial demand due to its immediate application as an ecofriendly, biodegradable biopesticide and also due to its various other significant bioactivities. To date, the only commercially feasible way to produce azadirachtin is extraction from seeds, but their availability is very limited as the tree flowers only once a year and only one-third of the fruits are collected due to operational problems. Further, due to the strict out-breeding nature of the plant, the seeds are highly heterozygous, resulting in inconsistent metabolite production. Therefore, in the present study, to achieve sustainable production of azadirachtin, dedifferentiated and redifferentiated calli derived from various explants of neem—zygotic embryo, leaf and ovary—were investigated for their potential to biosynthesize azadirachtin. High-performance liquid chromatography analysis of the in vitro cell lines showed the presence of azadirachtin in all the samples tested, the content of which in cultured cells varied with explant source and cell differentiation response. The presence of azadirachtin in samples was further confirmed by positive electrospray ionization mass spectroscopy. The zygotic embryo cultures of neem accumulated much higher amounts of azadirachtin than leaf and ovary cultures. Furthermore, organized in vitro callus cultures (redifferentiated) supported higher azadirachtin biosynthesis, while unorganized callus cultures (dedifferentiated) supported the least. The maximum azadirachtin content of 2.33 mg g−1 dry weight was obtained from redifferentiated immature zygotic embryo cultures.

Fibro/Adipogenic Progenitors (FAPs): Isolation by FACS and Culture.

PubMed

Low, Marcela; Eisner, Christine; Rossi, Fabio

2017-01-01

Fibro/adipogenic progenitors (FAPs ) are tissue-resident mesenchymal stromal cells (MSCs). Current literature supports a role for these cells in the homeostasis and repair of multiple tissues suggesting that FAPs may have extensive therapeutic potential in the treatment of numerous diseases. In this context, it is crucial to establish efficient and reproducible procedures to purify FAP populations from various tissues. Here, we describe a protocol for the isolation and cell culture of FAPs from murine skeletal muscle using fluorescence -activated cell sorting (FACS), which is particularly useful for experiments where high cell purity is an essential requirement. Identification, isolation, and cell culture of FAPs represent powerful tools that will help us to understand the role of these cells in different conditions and facilitate the development of safe and effective new treatments for diseases.

Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

PubMed Central

Lamb, Rebecca; Ambler, Carrie A.

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification. PMID:23326335

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids

PubMed Central

Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A.; Falvo D’Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

2016-01-01

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

PubMed Central

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells. PMID:26882313

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

PubMed

Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

2016-01-01

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture.

PubMed

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.

Mycoplasmal Infections Prevent Apoptosis and Induce Malignant Transformation of Interleukin-3-Dependent 32D Hematopoietic Cells

PubMed Central

Feng, Shaw-Huey; Tsai, Shien; Rodriguez, Jose; Lo, Shyh-Ching

1999-01-01

32D cells, a murine myeloid cell line, rapidly undergo apoptosis upon withdrawal of interleukin-3 (IL-3) supplement in culture. We found that 32D cells, if infected by several species of human mycoplasmas that rapidly activated NF-κB, would live and continue to grow in IL-3-depleted culture. Mycoplasma-infected cells showed no evidence of autocrine production of IL-3. Pyrrolidine dithiocarbamate (PDTC) blocked activation of NF-κB and led to prominent cell death. Heat-killed mycoplasmas or mycoplasmal membrane preparations alone could support continued growth of 32D cells in culture without IL-3 supplement for a substantial period of time. However, upon removal of heat-inactivated mycoplasmas, 32D cells quickly became apoptotic. In comparison, live Mycoplasma fermentans or M. penetrans infection for 4 to 5 weeks induced malignant transformation of 32D cells. Transformed 32D cells grew autonomously and no longer required support of growth-stimulating factors including IL-3 and mycoplasmas. The transformed 32D cells quickly formed tumors when injected into nude mice. Karyotyping showed that development of chromosomal changes and trisomy 19 was often associated with malignant transformation and tumorigenicity of 32D cells. Mycoplasmal infections apparently affected the fidelity of genomic transmission in cell division as well as checkpoints coordinating the progression of cell cycle events. PMID:10567525

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

PubMed

Sharma, Manjinder; Dubey, Pawan K; Kumar, Rajesh; Nath, Amar; Kumar, G Sai; Sharma, G Taru

2013-05-01

Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow-Derived Mouse Mesenchymal Stem Cells via a p53 Dependent Mechanism: Implications for Long-Term Culture Expansion

PubMed Central

Boregowda, Siddaraju; Krishnappa, Veena; Chambers, Jeremy; LoGrasso, Phillip V.; Lai, Wen-Tzu; Ortiz, Luis A.; Phinney, Donald G.

2013-01-01

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A and BAX expression and mitochondrial ROS generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immuno-depletion, and supported sustained MSC growth resulting in a 2300-fold increase in cumulative cell yield by 4th passage. MSCs cultured in 5% oxygen also exhibited enhanced tri-lineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA mediated knockdown of p53 in wild type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro. PMID:22367737

Organizational behavior of human umbilical vein endothelial cells

PubMed Central

1982-01-01

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization. PMID:6813338

Optimized suspension culture: the rotating-wall vessel

NASA Technical Reports Server (NTRS)

Hammond, T. G.; Hammond, J. M.

2001-01-01

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

PubMed Central

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-01-01

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.

PubMed

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A; Munusamy, Murugan A; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-03-23

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression.

PubMed

Ding, Wei; Nowakowski, Grzegorz S; Knox, Traci R; Boysen, Justin C; Maas, Mary L; Schwager, Susan M; Wu, Wenting; Wellik, Linda E; Dietz, Allan B; Ghosh, Asish K; Secreto, Charla R; Medina, Kay L; Shanafelt, Tait D; Zent, Clive S; Call, Timothy G; Kay, Neil E

2009-11-01

It was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B-cells was found in the co-culture. CD71 upregulation was more significantly associated with high-risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B-cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi-directional activation between bone marrow stromal cells and CLL B-cells.

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

PubMed

Rüger, Beate M; Buchacher, Tanja; Giurea, Alexander; Kubista, Bernd; Fischer, Michael B; Breuss, Johannes M

2018-01-01

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix. Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro , and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model. Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA. Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45 + leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures. Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.

Cell death in Tetrahymena thermophila: new observations on culture conditions.

PubMed

Christensen, S T; Sørensen, H; Beyer, N H; Kristiansen, K; Rasmussen, L; Rasmussen, M I

2001-01-01

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems. Copyright 2001 Academic Press.

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

PubMed

Liste-Calleja, Leticia; Lecina, Martí; Cairó, Jordi Joan

2014-04-01

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

PubMed Central

Catakovic, Kemal; Gassner, Franz Josef; Ratswohl, Christoph; Zaborsky, Nadja; Rebhandl, Stefan; Schubert, Maria; Steiner, Markus; Gutjahr, Julia Christine; Pleyer, Lisa; Egle, Alexander; Hartmann, Tanja Nicole; Greil, Richard; Geisberger, Roland

2018-01-01

ABSTRACT While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment. PMID:29296521

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

NASA Astrophysics Data System (ADS)

Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

2014-03-01

Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

The Effects of Hemodynamic Shear Stress on Stemness of Acute Myelogenous Leukemia (AML)

NASA Astrophysics Data System (ADS)

Raddatz, Andrew; Triantafillu, Ursula; Kim, Yonghyun (John)

2015-11-01

Cancer stem cells (CSCs) have recently been identified as the root cause of tumors generated from cancer cell populations. This is because these CSCs are drug-resistant and have the ability to self-renew and differentiate. Current methods of culturing CSCs require much time and money, so cancer cell culture protocols, which maximize yield of CSCs are needed. It was hypothesized that the quantity of Acute myelogenous leukemia stem cells (LSCs) would increase after applying shear stress to the leukemia cells based on previous studies with breast cancer in bioreactors. The shear stress was applied by pumping the cells through narrow tubing to mimic the in vivo bloodstream environment. In support of the hypothesis, shear stress was found to increase the amount of LSCs in a given leukemia population. This work was supported by NSF REU Site Award 1358991.

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

PubMed

Okeyo, Kennedy Omondi; Kurosawa, Osamu; Yamazaki, Satoshi; Oana, Hidehiro; Kotera, Hidetoshi; Nakauchi, Hiromitsu; Washizu, Masao

2015-10-01

Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 μm in pitch) with narrow mesh strands (3-5 μm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Kakeda, M; Yoneya, T; Tsumura, H; Ohashi, H; Yamaguchi, Y; Motoki, K; Sudo, T; Mori, K J

1996-09-01

The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.

Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

PubMed Central

Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

2015-01-01

ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

Culture on electrospun polyurethane scaffolds decreases atrial natriuretic peptide expression by cardiomyocytes in vitro.

PubMed

Rockwood, Danielle N; Akins, Robert E; Parrag, Ian C; Woodhouse, Kimberly A; Rabolt, John F

2008-12-01

The function of the mammalian heart depends on the functional alignment of cardiomyocytes, and controlling cell alignment is an important consideration in biomaterial design for cardiac tissue engineering and research. The physical cues that guide functional cell alignment in vitro and the impact of substrate-imposed alignment on cell phenotype, however, are only partially understood. In this report, primary cardiac ventricular cells were grown on electrospun, biodegradable polyurethane (ES-PU) with either aligned or unaligned microfibers. ES-PU scaffolds supported high-density cultures and cell subpopulations remained intact over two weeks in culture. ES-PU cultures contained electrically-coupled cardiomyocytes with connexin-43 localized to points of cell:cell contact. Multi-cellular organization correlated with microfiber orientation and aligned materials yielded highly oriented cardiomyocyte groupings. Atrial natriuretic peptide, a molecular marker that shows decreasing expression during ventricular cell maturation, was significantly lower in cultures grown on ES-PU scaffolds than in those grown on tissue culture polystyrene. Cells grown on aligned ES-PU had significantly lower steady state levels of ANP and constitutively released less ANP over time indicating that scaffold-imposed cell organization resulted in a shift in cell phenotype to a more mature state. We conclude that the physical organization of microfibers in ES-PU scaffolds impacts both multi-cellular architecture and cardiac cell phenotype in vitro.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

Increased NIH 3T3 fibroblast functions on cell culture dishes which mimic the nanometer fibers of natural tissues.

PubMed

Bhardwaj, Garima; Webster, Thomas J

2015-01-01

Traditional flat tissue cell culture dishes have consisted of polystyrene treated with plasma gases for growing, subculturing, and studying cell behavior in vitro. However, increasingly it has been observed that mimicking natural tissue properties (such as chemistry, three-dimensional structure, mechanical properties, etc) in vitro can lead to a better correlation of in vitro to in vivo cellular functions. The following studies compared traditional NIH 3T3 fibroblasts' functions on XanoMatrix scaffolds to standard tissue culture polystyrene. Results found significantly greater fibroblast adhesion and proliferation on XanoMatrix cell culture dishes which mimic the nanoscale geometry of natural tissue fibers with true, tortuous fiber beds creating a robust, consistent, and versatile growth platform. In this manner, this study supports that cell culture dishes which mimic features of natural tissues should be continually studied for a wide range of applications in which mimicking natural cellular functions are important.

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

PubMed

Kariya, Yoshinobu; Sato, Hiroki; Katou, Naoko; Kariya, Yukiko; Miyazaki, Kaoru

2012-01-01

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.

Sponge-supported cultures of primary head and neck tumors for an optimized preclinical model.

PubMed

Dohmen, Amy J C; Sanders, Joyce; Canisius, Sander; Jordanova, Ekaterina S; Aalbersberg, Else A; van den Brekel, Michiel W M; Neefjes, Jacques; Zuur, Charlotte L

2018-05-18

Treatment of advanced head and neck cancer is associated with low survival, high toxicity and a widely divergent individual response. The sponge-gel-supported histoculture model was previously developed to serve as a preclinical model for predicting individual treatment responses. We aimed to optimize the sponge-gel-supported histoculture model and provide more insight in cell specific behaviour by evaluating the tumor and its microenvironment using immunohistochemistry. We collected fresh tumor biopsies from 72 untreated patients and cultured them for 7 days. Biopsies from 57 patients (79%) were successfully cultured and 1451 tumor fragments (95.4%) were evaluated. Fragments were scored for percentage of tumor, tumor viability and proliferation, EGF-receptor expression and presence of T-cells and macrophages. Median tumor percentage increased from 53% at day 0 to 80% at day 7. Viability and proliferation decreased after 7 days, from 90% to 30% and from 30% to 10%, respectively. Addition of EGF, folic acid and hydrocortisone can lead to improved viability and proliferation, however this was not systematically observed. No patient subgroup could be identified with higher culture success rates. Immune cells were still present at day 7, illustrating that the tumor microenvironment is sustained. EGF supplementation did not increase viability and proliferation in patients overexpressing EGF-Receptor.

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.

PubMed

Sokol, Ethan S; Miller, Daniel H; Breggia, Anne; Spencer, Kevin C; Arendt, Lisa M; Gupta, Piyush B

2016-03-01

Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.

Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array.

PubMed

Kreutzer, Joose; Ylä-Outinen, Laura; Mäki, Antti-Juhana; Ristola, Mervi; Narkilahti, Susanna; Kallio, Pasi

2017-03-15

Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO 2 supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential. We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber. We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments. Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO 2 gas. Utilizing dry gas supply makes the proposed chamber simple to use. Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Image analysis supported moss cell disruption in photo-bioreactors.

PubMed

Lucumi, A; Posten, C; Pons, M-N

2005-05-01

Diverse methods for the disruption of cell entanglements and pellets of the moss Physcomitrella patens were tested in order to improve the homogeneity of suspension cultures. The morphological characterization of the moss was carried out by means of image analysis. Selected morphological parameters were defined and compared to the reduction of the carbon dioxide fixation, and the released pigments after cell disruption. The size control of the moss entanglements based on the rotor stator principle allowed a focused shear stress, avoiding a severe reduction in the photosynthesis. Batch cultures of P. patens in a 30.0-l pilot tubular photo-bioreactor with cell disruption showed no significant variation in growth rate and a delayed cell differentiation, when compared to undisrupted cultures. A highly controlled photoautotrophic culture of P. patens in a scalable photo-bioreactor was established, contributing to the development required for the future use of mosses as producers of relevant heterologous proteins.

Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells.

PubMed

White, Patricia M; Doetzlhofer, Angelika; Lee, Yun Shain; Groves, Andrew K; Segil, Neil

2006-06-22

Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.

Tuning differentiation signals for efficient propagation and in vitro validation of rat embryonic stem cell cultures.

PubMed

Meek, Stephen; Sutherland, Linda; Burdon, Tom

2015-01-01

The rat is one of the most commonly used laboratory animals in biomedical research and the recent isolation of genuine pluripotent rat embryonic stem (ES) cell lines has provided new opportunities for applying contemporary genetic engineering techniques to the rat and enhancing the use of this rodent in scientific research. Technical refinements that improve the stability of the rat ES cell cultures will undoubtedly further strengthen and broaden the use of these stem cells in biomedical research. Here, we describe a relatively simple and robust protocol that supports the propagation of germ line competent rat ES cells, and outline how tuning stem cell signaling using small molecule inhibitors can be used to both stabilize self-renewal of rat ES cell cultures and aid evaluation of their differentiation potential in vitro.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

PubMed

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

PubMed

Mandatori, Domitilla; Penolazzi, Letizia; Pipino, Caterina; Di Tomo, Pamela; Di Silvestre, Sara; Di Pietro, Natalia; Trevisani, Sara; Angelozzi, Marco; Ucci, Mariangela; Piva, Roberta; Pandolfi, Assunta

2018-02-01

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment. Copyright © 2017 John Wiley & Sons, Ltd.

Improved Performance in Mammalian Cell Perfusion Cultures by Growth Inhibition.

PubMed

Wolf, Moritz K F; Closet, Aurélie; Bzowska, Monika; Bielser, Jean-Marc; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

2018-05-21

Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Restrictions in Cell Cycle Progression of Adult Vestibular Supporting Cells in Response to Ectopic Cyclin D1 Expression

PubMed Central

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H.; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells. PMID:22073316

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

PubMed

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.

Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture.

PubMed

Shaikh, Ambreen; Anand, Sandhya; Kapoor, Sona; Ganguly, Ranita; Bhartiya, Deepa

2017-04-01

Very small embryonic-like stem cells (VSELs) have been reported in various adult tissues, express pluripotent and primordial germ cells (PGCs) specific markers, are mobilized under stress/disease conditions, give rise to tissue committed progenitors and thus help regenerate and maintain homeostasis. The aim of the present study was to evaluate in vitro differentiation potential of VSELs using a quantitative approach. VSELs were collected from mouse bone marrow after 4 days of 5-fluorouracil (5-FU, 150 mg/Kg) treatment, further enriched by size based filtration and cultured on a feeder support in the presence of specific differentiation media. Cultured VSELs were found to differentiate into all three embryonic germ cell lineages, germ and hematopoietic cells after 14 days in culture. This was confirmed by studying Nestin, PDX-1, NKX2.5, DAZL, CD45 and other markers expression by various approaches. Very small, CD45 negative cells collected and enriched from GFP positive 5-FU treated mice bone marrow transitioned into CD45 positive cells in vitro thus demonstrating that VSELs can give rise to hematopoietic stem cells (HSCs). We envision that VSELs may be responsible for plasticity and ability of bone marrow cells to give rise to non-hematopoietic tissue progenitors of all 3 germ layers. Moreover the ability of VSELs to differentiate into germ cells as well as all the three lineages provides further evidence to support their pluripotent state and confirms developmental link between bone marrow VSELs and PGCs. The property of quiescence, no risk of teratoma formation and autologus source, make pluripotent VSELs a potential candidate to facilitate endogenous regeneration compared to cell replacement strategy envisioned using embryonic and induced pluripotent stem cells.

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

Plasma modified PLA electrospun membranes for actinorhodin production intensification in Streptomyces coelicolor immobilized-cell cultivations.

PubMed

Scaffaro, Roberto; Lopresti, Francesco; Sutera, Alberto; Botta, Luigi; Fontana, Rosa Maria; Gallo, Giuseppe

2017-09-01

Most of industrially relevant bioproducts are produced by submerged cultivations of actinomycetes. The immobilization of these Gram-positive filamentous bacteria on suitable porous supports may prevent mycelial cell-cell aggregation and pellet formation which usually negatively affect actinomycete submerged cultivations, thus, resulting in an improved biosynthetic capability. In this work, electrospun polylactic acid (PLA) membranes, subjected or not to O 2 -plasma treatment (PLA-plasma), were used as support for immobilized-cell submerged cultivations of Streptomyces coelicolor M145. This strain produces different bioactive compounds, including the blue-pigmented actinorhodin (ACT) and red-pigmented undecylprodigiosin (RED), and constitutes a model for the study of antibiotic-producing actinomycetes. Wet contact angles and X-ray photoelectron spectroscopy analysis confirmed the increased wettability of PLA-plasma due to the formation of polar functional groups such as carboxyl and hydroxyl moieties. Scanning electron microscope observations, carried out at different incubation times, revealed that S. coelicolor immobilized-cells created a dense "biofilm-like" mycelial network on both kinds of PLA membranes. Cultures of S. coelicolor immobilized-cells on PLA or PLA-plasma membranes produced higher biomass (between 1.5 and 2 fold) as well as higher levels of RED and ACT than planktonic cultures. In particular, cultures of immobilized-cells on PLA and PLA-plasma produced comparable levels of RED that were approximatively 4 and 5 fold higher than those produced by planktonic cultures, respectively. In contrast, levels of ACT produced by immobilized-cell cultures on PLA and PLA-plasma were different, being 5 and 10 fold higher than those of planktonic cultures, respectively. Therefore, this is study demonstrated the positive influence of PLA membrane on growth and secondary metabolite production in S. coelicolor and also revealed that O 2 -plasma treated PLA membranes specifically promoted higher ACT production than not treated membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

3D culture of Her2+ breast cancer cells promotes AKT to MAPK switching and a loss of therapeutic response.

PubMed

Gangadhara, Sharath; Smith, Chris; Barrett-Lee, Peter; Hiscox, Stephen

2016-06-01

The Her2 receptor is overexpressed in up to 25 % of breast cancers and is associated with a poor prognosis. Around half of Her2+ breast cancers also express the estrogen receptor and treatment for such tumours can involve both endocrine and Her2-targeted therapies. However, despite preclinical data supporting the effectiveness of these agents, responses can vary widely in the clinical setting. In light of the increasing evidence pointing to the interplay between the tumour and its extracellular microenvironment as a significant determinant of therapeutic sensitivity and response here we investigated the impact of 3D matrix culture of breast cancer cells on their therapeutic sensitivity. A 3D Matrigel-based culture system was established and optimized for the growth of ER+/Her2+ breast cancer cell models. Growth of cells in response to trastuzumab and endocrine agents in 3D culture versus routine monolayer culture were assessed using cell counting and Ki67 staining. Endogenous and trastuzumab-modulated signalling pathway activity in 2D and 3D cultures were assessed using Western blotting. Breast cancer cells in 3D culture displayed an attenuated response to both endocrine agents and trastuzumab compared with cells cultured in traditional 2D monolayers. Underlying this phenomenon was an apparent matrix-induced shift from AKT to MAPK signalling; consequently, suppression of MAPK in 3D cultures restores therapeutic response. These data suggest that breast cancer cells in 3D culture display a reduced sensitivity to therapeutic agents which may be mediated by internal MAPK-mediated signalling. Targeting of adaptive pathways that maintain growth in 3D culture may represent an effective strategy to improve therapeutic response clinically.

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate.

PubMed

Shen, Chun Fang; Jacob, Danielle; Zhu, Tao; Bernier, Alice; Shao, Zhongqi; Yu, Xuefeng; Patel, Mehul; Lanthier, Stephane; Kamen, Amine

2016-06-17

Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition

PubMed Central

Mahoney, Mark M.; Staley, Kevin J.

2017-01-01

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy. PMID:28225808

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

PubMed

Liu, Jing; Saponjian, Yero; Mahoney, Mark M; Staley, Kevin J; Berdichevsky, Yevgeny

2017-01-01

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Yoneya, T; Kakeda, M; Tsumura, H; Ohashi, H; Mori, K J

1997-04-01

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

KDR (VEGFR2) identifies a conserved human and murine hepatic progenitor and instructs early liver development

PubMed Central

Goldman, Orit; Han, Songyan; Sourrisseau, Marion; Dziedzic, Noelle; Hamou, Wissam; Corneo, Barbara; D’Souza, Sunita; Sato, Thomas; Kotton, Darrell N.; Bissig, Karl-Dimiter; Kalir, Tamara; Jacobs, Adam; Evans, Todd; Evans, Matthew J.; Gouon-Evans, Valerie

2013-01-01

SUMMARY Understanding the fetal hepatic niche is essential for optimizing the generation of functional hepatocyte-like (hepatic) cells from human embryonic stem cells (hESCs). Here, we show that KDR (VEGFR2), previously assumed to be mostly restricted to mesodermal lineages, marks a hESC-derived hepatic progenitor. hESC-derived endoderm cells do not express KDR, but when cultured in media supporting hepatic differentiation, generate KDR+ hepatic progenitors and KDR- hepatic cells. KDR+ progenitors require active KDR signaling both to instruct their own differentiation into hepatic cells, and to support non-cell-autonomously the functional maturation of co-cultured KDR- hepatic cells. Analysis of human fetal livers suggests that similar progenitors are present in human livers. Lineage tracing in mice provides in vivo evidence of a KDR+ hepatic progenitor for fetal hepatoblasts and subsequently adult hepatocytes and cholangiocytes. Altogether, our findings reveal that KDR is a conserved marker for endoderm-derived hepatic progenitors, and a functional receptor instructing early liver development. PMID:23746980

Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineering.

PubMed

Donegan, Gail C; Hunt, John A; Rhodes, Nicholas

2010-02-01

Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space.

Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Fenxi, E-mail: fxzhang0824@gmail.com; Hong, Yan; Liang, Wenmei

Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neuralmore » stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.« less

Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

PubMed

Liew, Lawrence J; Day, Richard M; Dilley, Rodney J

2017-03-01

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

PubMed Central

Iwamaru, Yoshifumi; Takenouchi, Takato; Imamura, Morikazu; Shimizu, Yoshihisa; Miyazawa, Kohtaro; Mohri, Shirou; Yokoyama, Takashi

2013-01-01

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level. PMID:23740992

Dormant Cells of Staphylococcus aureus Are Resuscitated by Spent Culture Supernatant

PubMed Central

Pascoe, Ben; Dams, Lucy; Wilkinson, Tom S.; Harris, Llinos G.; Bodger, Owen; Mack, Dietrich; Davies, Angharad P.

2014-01-01

We describe the first in vitro model of dormancy in Staphylococcus aureus, showing that cells are generated which can be resuscitated by addition of spent medium supernatant taken from cultures of the same organism. Over 30 days, culturable counts in dormant cultures of S. aureus SH1000 fell from 106–107 cfu/ml to <10 cfu/ml as measured by the Most Probable Number method in liquid culture, while total counts as determined by microscopy, and supported by data from RT-qPCR, remained around 106–107 cells/ml. Supplementing cultures with 25–50% spent medium resulted in a >600-fold increase in bacterial growth. Resuscitation was a specific effect, greatly reduced by boiling or addition of trypsin to the spent supernatant. Supernatant also effected a reduction in lag phase of dormant cultures. SEM demonstrated the presence of small coccoid cells in dormant cultures. The results are similar to those seen with resuscitation promoting factors (Rpfs) in actinobacteria. This is the first time resuscitation has been demonstrated in Staphylococcus aureus, which is an important human pathogen. A better understanding of control and reactivation of dormant cells could lead to major improvements in managing staphylococcal infections; resuscitation could be an important step in restoring susceptibility to antibiotic treatment. PMID:24523858

21st Century Cell Culture for 21st Century Toxicology.

PubMed

Pamies, David; Hartung, Thomas

2017-01-17

There is no good science in bad models. Cell culture is especially prone to artifacts. A number of novel cell culture technologies have become more broadly available in the 21st century, which allow overcoming limitations of traditional culture and are more physiologically relevant. These include the use of stem-cell derived human cells, cocultures of different cell types, scaffolds and extracellular matrices, perfusion platforms (such as microfluidics), 3D culture, organ-on-chip technologies, tissue architecture, and organ functionality. The physiological relevance of such models is further enhanced by the measurement of biomarkers (e.g., key events of pathways), organ specific functionality, and more comprehensive assessment cell responses by high-content methods. These approaches are still rarely combined to create microphysiological systems. The complexity of the combination of these technologies can generate results closer to the in vivo situation but increases the number of parameters to control, bringing some new challenges. In fact, we do not argue that all cell culture needs to be that sophisticated. The efforts taken are determined by the purpose of our experiments and tests. If only a very specific molecular target to cell response is of interest, a very simple model, which reflects this, might be much more suited to allow standardization and high-throughput. However, the less defined the end point of interest and cellular response are, the better we should approximate organ- or tissue-like culture conditions to make physiological responses more probable. Besides these technologic advances, important progress in the quality assurance and reporting on cell cultures as well as the validation of cellular test systems brings the utility of cell cultures to a new level. The advancement and broader implementation of Good Cell Culture Practice (GCCP) is key here. In toxicology, this is a major prerequisite for meaningful and reliable results, ultimately supporting risk assessment and product development decisions.

Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

PubMed

Kim, Ha Ryong; Cho, Han Soo; Shin, Da Young; Chung, Kyu Hyuck

2017-02-01

In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis.

PubMed

Touil, Hanane; Kobert, Antonia; Lebeurrier, Nathalie; Rieger, Aja; Saikali, Philippe; Lambert, Caroline; Fawaz, Lama; Moore, Craig S; Prat, Alexandre; Gommerman, Jennifer; Antel, Jack P; Itoyama, Yasuto; Nakashima, Ichiro; Bar-Or, Amit

2018-04-19

The success of clinical trials of selective B cell depletion in patients with relapsing multiple sclerosis (MS) indicates B cells are important contributors to peripheral immune responses involved in the development of new relapses. Such B cell contribution to peripheral inflammation likely involves antibody-independent mechanisms. Of growing interest is the potential that B cells, within the MS central nervous system (CNS), may also contribute to the propagation of CNS-compartmentalized inflammation in progressive (non-relapsing) disease. B cells are known to persist in the inflamed MS CNS and are more recently described as concentrated in meningeal immune-cell aggregates, adjacent to the subpial cortical injury which has been associated with progressive disease. How B cells are fostered within the MS CNS and how they may contribute locally to the propagation of CNS-compartmentalized inflammation remain to be elucidated. We considered whether activated human astrocytes might contribute to B cell survival and function through soluble factors. B cells from healthy controls (HC) and untreated MS patients were exposed to primary human astrocytes that were either maintained under basal culture conditions (non-activated) or pre-activated with standard inflammatory signals. B cell exposure to astrocytes included direct co-culture, co-culture in transwells, or exposure to astrocyte-conditioned medium. Following the different exposures, B cell survival and expression of T cell co-stimulatory molecules were assessed by flow cytometry, as was the ability of differentially exposed B cells to induce activation of allogeneic T cells. Secreted factors from both non-activated and activated human astrocytes robustly supported human B cell survival. Soluble products of pre-activated astrocytes also induced B cell upregulation of antigen-presenting cell machinery, and these B cells, in turn, were more efficient activators of T cells. Astrocyte-soluble factors could support survival and activation of B cell subsets implicated in MS, including memory B cells from patients with both relapsing and progressive forms of disease. Our findings point to a potential mechanism whereby activated astrocytes in the inflamed MS CNS not only promote a B cell fostering environment, but also actively support the ability of B cells to contribute to the propagation of CNS-compartmentalized inflammation, now thought to play key roles in progressive disease.

Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation.

PubMed

Marrazzo, Pasquale; Paduano, Francesco; Palmieri, Francesca; Marrelli, Massimo; Tatullo, Marco

2016-01-01

Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H 2 O 2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

PubMed Central

Palmieri, Francesca; Marrelli, Massimo

2016-01-01

Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use. PMID:27774106

Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

PubMed

Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system.

Cisplatin ototoxicity blocks sensory regeneration in the avian inner ear.

PubMed

Slattery, Eric L; Warchol, Mark E

2010-03-03

Cisplatin is a chemotherapeutic agent that is widely used in the treatment of solid tumors. Ototoxicity is a common side effect of cisplatin therapy and often leads to permanent hearing loss. The sensory organs of the avian ear are able to regenerate hair cells after aminoglycoside ototoxicity. This regenerative response is mediated by supporting cells, which serve as precursors to replacement hair cells. Given the antimitotic properties of cisplatin, we examined whether the avian ear was also capable of regeneration after cisplatin ototoxicity. Using cell and organ cultures of the chick cochlea and utricle, we found that cisplatin treatment caused apoptosis of both auditory and vestibular hair cells. Hair cell death in the cochlea occurred in a unique pattern, progressing from the low-frequency (distal) region toward the high-frequency (proximal) region. We also found that cisplatin caused a dose-dependent reduction in the proliferation of cultured supporting cells as well as increased apoptosis in those cells. As a result, we observed no recovery of hair cells after ototoxic injury caused by cisplatin. Finally, we explored the potential for nonmitotic hair cell recovery via activation of Notch pathway signaling. Treatment with the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester failed to promote the direct transdifferentiation of supporting cells into hair cells in cisplatin-treated utricles. Taken together, our data show that cisplatin treatment causes maintained changes to inner ear supporting cells and severely impairs the ability of the avian ear to regenerate either via proliferation or by direct transdifferentiation.

Effects of simulated microgravity on mouse Sertoli cells in culture

NASA Astrophysics Data System (ADS)

Angela, Masini Maria; Prato, Paola; Linda, Scarabelli; Lanza, Cristina; Palmero, Silvio; Pointis, Georges; Ricci, Franco; Strollo, Felice

With the advent of space flights questions concerning the effects of microgravity (0xG) on hu-man reproduction physiology have got priority Spermatogenesis is a complex, highly ordered process of cell division and differentiation by which spermatogonial cells give rise to mature spermatozoa. Sertoli cells play a crucial role in the development of germ cells and the regulation of spermatogenesis. In this study the influence of 0xG on Sertoli cells was evaluated. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal (using the 3D reconstruction generated from a stack of confocal images) and SHBG changes by immunohistochemistry, for antioxidant agents by RT-PCR and for culture medium lactate concentrations by wet chemistry. Cells were cultured for 6, 24 and 48 hrs on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1xG) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or RNA-extracted or used for culture medium lactate measurements as needed. At 0xG Sertoli cytoskeleton got disorganized, microtubules fragmented and SHBG undetectable already after 24 hrs, with alterations wors-ening further until 48 hrs; various antioxidant systems (SOD, GST, PARP, MTs) appreciably increased during the first 24 hrs but significantly decreased at 48 hrs. No changes occurred in 1xG samples. At least initially, 0xG seems to perturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0xG slightly decreased only after 24 hrs. Further experiments need to be carried out in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out eventually pending male infertility consequences, which would be a problem nowadays, when life expectancy increases and male fertility might become a social issue often extending into 60 years and over. (experiment funded by ASI, through a grant within the OSMA project).

Xenotransplantation of neonatal porcine liver cells.

PubMed

Garkavenko, O; Emerich, D F; Muzina, M; Muzina, Z; Vasconcellos, A V; Ferguson, A B; Cooper, I J; Elliott, R B

2005-01-01

Xenotransplantation of porcine liver cell types may provide a means of overcoming the shortage of suitable donor tissues to treat hepatic diseases characterized by inherited inborn errors of metabolism or protein production. Here we report the successful isolation, culture, and xenotransplantation of liver cells harvested from 7- to 10-day-old piglets. Liver cells were isolated and cultured immediately after harvesting. Cell viability was excellent (>90%) over the duration of the in vitro studies (3 weeks) and the cultured cells continued to significantly proliferate. These cells also retained their normal secretory and metabolic capabilities as determined by continued release of albumin, factor 8, and indocyanin green (ICG) uptake. After 3 weeks in culture, porcine liver cells were loaded into immunoisolatory macro devices (Theracyte devices) and placed into the intraperitoneal cavity of immunocompetant CD1 mice. Eight weeks later, the devices were retrieved and the cells analyzed for posttransplant determinations of survival and function. Post mortem analysis confirmed that the cell-loaded devices were biocompatible, and were well-tolerated without inducing any notable inflammatory reaction in the tissues immediately surrounding the encapsulated cells. Finally, the encapsulated liver cells remained viable and functional as determined by histologic analyses and ICG uptake/release. The successful harvesting, culturing, and xenotransplantation of functional neonatal pig liver cells support the continued development of this approach for treating a range of currently undertreated or intractable hepatic diseases.

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

PubMed

Singh, Mahipal; Sharma, Anil K

2011-02-01

Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

PubMed Central

Neeper, I D; Patton, D L; Kuo, C C

1990-01-01

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

Microengineered embryonic stem cells niche to induce neural differentiation.

PubMed

Joshi, Ramila; Tavana, Hossein

2015-08-01

A major challenge in therapeutic use of embryonic stem cells (ESCs) for treating neurodegenerative diseases is creating a niche in vitro for controlled neural-specific differentiation of ESCs. We employ a niche microengineering approach to derive neural cells from ESCs by mimicking embryonic development in terms of direct intercellular interactions. Using a polymeric aqueous two-phase system (ATPS) microprinting technology, murine ESCs (mESCs) are precisely localized over a monolayer of supporting stromal cells to allow formation of individual mESC colonies. Polyethylene glycol (PEG) and dextran (DEX) are dissolved in culture media to form two immiscible aqueous solutions. A robotic liquid handler is used to print a nanoliter-volume drop of the denser DEX phase solution containing mESCs onto a confluent layer of supporting PA6 stromal cells submerged in the aqueous PEG phase. mESCs proliferate into isolated colonies of uniform size. For the first time, a comprehensive protein expression analysis of individual mESC colonies is performed over a two-week culture period to track temporal progression of cells from a pluripotent stage to specific neural cells. Starting from day 4, the expression of nestin, neural cell adhesion molecule (NCAM), and beta-III tubulin shows a significant increase but then levels off after the first week of culture. The expression of specific neural cell markers glial fibrillary acidic protein (GFAP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and tyrosine hydroxylase (TH) is elevated during the second week of culture. This microengineering approach to control ESCs differentiation niche combined with the time-course protein expression analysis of individual differentiating colonies facilitates understanding of evolution of specific neural cells from ESCs and identifying underlying molecular markers.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

PubMed

Cerino, Giulia; Gaudiello, Emanuele; Grussenmeyer, Thomas; Melly, Ludovic; Massai, Diana; Banfi, Andrea; Martin, Ivan; Eckstein, Friedrich; Grapow, Martin; Marsano, Anna

2016-01-01

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment. © 2015 Wiley Periodicals, Inc.

Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis.

PubMed

Wen, Qing; Li, Nan; Xiao, Xiang; Lui, Wing-Yee; Chu, Darren S; Wong, Chris K C; Lian, Qingquan; Ge, Renshan; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

2018-02-12

Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.

Graphene foam as a biocompatible scaffold for culturing human neurons

PubMed Central

Mattei, Cristiana; Nasr, Babak; Hudson, Emma J.; Alshawaf, Abdullah J.; Chana, Gursharan; Everall, Ian P.; Dottori, Mirella; Skafidas, Efstratios

2018-01-01

In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation. PMID:29657752

Basic FGF Support of Human Embryonic Stem Cell Self-Renewal

PubMed Central

Levenstein, Mark E.; Ludwig, Tenneille E.; Xu, Ren-He; Llanas, Rachel A.; VanDenHeuvel-Kramer, Kaitlyn; Manning, Daisy; Thomson, James A.

2015-01-01

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic FGF (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. Recently, it has been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Here we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100 and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture the cells formed teratomas when injected into SCID-beige mice, and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells, and suggest that fibroblasts and fibroblast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold. PMID:16282444

Adipose derived mesenchymal stem cells partially rescue mitomycin C treated ARPE19 cells from death in co-culture condition.

PubMed

Singh, Amar K; Srivastava, Girish K; García-Gutiérrez, María T; Pastor, J Carlos

2013-12-01

Age-related macular degeneration is a retinal disease with important damage at the RPE layer. This layer is considered a target for therapeutical approaches. Stem cell transplantation is a promising option for retinal diseases. Adipose derived mesenchymal stem cells secret growth factors which might play a significant role in RPE maintenance. This study aimed to evaluate human AD-MSCs ability to rescue mitomycin C treated dying ARPE19 cells in co-culture condition. ARPE19 cells were treated with MMC (50 μg/ml, 100 μg/ml and 200 μg/ml) for 2 hours to induce cell death. These treated cells were co-cultured with hAD-MSCs in indirect co-culture system for 3 days and 3 weeks. Then the viability, growth and proliferation of these ARPE19 cells were evaluated by a cell viability/cytotoxicity assay kit and Alamar Blue (AB) assay. Untreated ARPE19 cells and human skin fibroblasts (HSF) were used as controls. MMC blocked ARPE19 cell proliferation significantly in 3 days and cells were almost completely dead after 3 weeks. Cell toxicity of MMC increased significantly with concentration. When these cells were co-cultured with hAD-MSCs, a significant growth difference was observed in treated cells compared to untreated cells. hAD-MSCs rescue capacity was also significantly higher than HSF for treated ARPE19 cells. This study showed that hAD-MSCs rescued MMC treated ARPE19 cells from death. It probably occurred due to undefined growth factors secreted by hAD-MSCs in the medium, shared by treated ARPE19 cells in co-culture conditions. This study supports further evaluation of the effect of hAD-MSCs subretinal transplantation over the RPE degeneration process in AMD patients.

Injectable thermosensitive chitosan/β-glycerophosphate/collagen hydrogel maintains the plasticity of skeletal muscle satellite cells and supports their in vivo viability.

PubMed

Ding, Ke; Yang, Zhong; Zhang, Yu-Long; Xu, Jian-Zhong

2013-09-01

A cell carrier plays an important role in the maintenance, growth and engraftment of specific cells aimed for defined therapeutic uses in many tissue engineering strategies. A suitable microenvironment for the cells allows for the maximum efficacy of the hybrid device. We have prepared an injectable thermosensitive chitosan/β-glycerophosphate/collagen (C/GP/Co) gel and investigated its potential application as a support for the culture of skeletal muscle satellite cells (SMSCs). A cell viability assay was used to evaluate the in vitro cytocompatibility of the gel. Cell growth was assessed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and histological analysis. The influence of the C/GP/Co gel on the plasticity of SMSCs seeded at the surface of the gel was assessed by induction of the myogenic, osteogenic and adipogenic differentiation. C/GP/Co gel provided the appropriate environment for the culture of SMSCs in vitro. In addition, the C/GP/Co gel supported SMSC plasticity. In vivo testing of the SMSC-seeded gel was investigated by subcutaneous injection into the dorsum of nude mice. Cell viability was assessed both by in vivo imaging and histological examination of the explants. In conclusion, C/GP/Co hydrogel is a cytocompatible carrier for the in vivo delivery of SMSCs and supportive for SMSC plasticity. Thus, this gel has potential applications in tissue engineering and regenerative medicine. © 2013 International Federation for Cell Biology.

Continuous microcarrier-based cell culture in a benchtop microfluidic bioreactor.

PubMed

Abeille, F; Mittler, F; Obeid, P; Huet, M; Kermarrec, F; Dolega, M E; Navarro, F; Pouteau, P; Icard, B; Gidrol, X; Agache, V; Picollet-D'hahan, N

2014-09-21

Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.

Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

PubMed

Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

2015-05-21

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.

Multizone Paper Platform for 3D Cell Cultures

PubMed Central

Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

2011-01-01

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

Combined platelet and plasma derivatives enhance proliferation of stem/progenitor cells maintaining their differentiation potential.

PubMed

Muraglia, Anita; Todeschi, Maria Rosa; Papait, Andrea; Poggi, Alessandro; Spanò, Raffaele; Strada, Paolo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2015-12-01

Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Studies on the apoptotic activity of natural and synthetic retinoids: discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apoptosis in neuronal and HL-60 cells.

PubMed

Simoni, Daniele; Giannini, Giuseppe; Roberti, Marinella; Rondanin, Riccardo; Baruchello, Riccardo; Rossi, Marcello; Grisolia, Giuseppina; Invidiata, Francesco Paolo; Aiello, Stefania; Marino, Silvia; Cavallini, Sabrina; Siniscalchi, Anna; Gebbia, Nicola; Crosta, Lucia; Grimaudo, Stefania; Abbadessa, Vincenzo; Di Cristina, Antonietta; Tolomeo, Manlio

2005-06-30

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells.

PubMed

Garba, Abubakar; Acar, Delphine D; Roukaerts, Inge D M; Desmarets, Lowiese M B; Devriendt, Bert; Nauwynck, Hans J

2017-09-01

Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell cultures were successfully established and characterized and they supported the proliferation of red bone marrow hematopoietic cells, which finally differentiated into monocytic cells and CD4 + and CD8 + cells. Copyright © 2017. Published by Elsevier B.V.

Apple derived cellulose scaffolds for 3D mammalian cell culture.

PubMed

Modulevsky, Daniel J; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

PubMed Central

Modulevsky, Daniel J.; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E.

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment. PMID:24842603

Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

PubMed

Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

2014-10-01

Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

Insufficient interleukin-2 production from splenic CD4+ T cells causes impaired cell proliferation and early apoptosis in SAMP1, a strain of senescence-accelerated mouse

PubMed Central

Nishimura, Yasumitsu; Hosokawa, Tomohide; Hosono, Masamichi; Baba, Mitsuo; Hosokawa, Masanori

2002-01-01

We examined the proliferative and cytokine-producing activities of CD4+ T cells from young mice of the senescence-accelerated mouse strain SAMP1, which had shown markedly low T-dependent antibody-producing responses. When splenic T cells were cultured with concanavalin A (Con A), the percentage of CD4+ cells decreased earlier in SAMP1 than in C3H/He mice. At 40 hr of culture, the percentage of BrdU-labelled proliferating CD4+ cells increased strongly in C3H/He, but only slightly in SAMP1. When purified CD4+ T cells were cultured with Con A, the percentage of 5-bromo-2′-deoxyuridine (BrdU)-labelled cells peaked at around 48 hr of culture in both strains, but decreased significantly at 64 hr in SAMP1. The production of interleukin (IL)-2 but not IL-4 or interferon-γ (IFN-γ) was significantly lower in SAMP1 than in C3H/He at 48 hr of culture. IL-2 production was also markedly low in SAMP1, even under the stimulation of anti-CD3 with anti-CD28 antibodies. The frequency of cells producing IL-2 was significantly lower in SAMP1 than in C3H/He at 6–24 hr of culture with Con A. The percentage of annexin-positive and propidium iodide (PI)-negative apoptotic cells was significantly higher in SAMP1 than in C3H/He at 96 hr of culture. Exogenous IL-2 prevented the decrease in BrdU-labelled cells and the increase in apoptotic cells in the SAMP1 cell culture. These results indicate that SAMP1 CD4+ T cells cannot produce IL-2 at levels sufficient to support cell proliferation and survival. This may account for the weak T-dependent antibody response in SAMP1 mice. PMID:12383198

A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

PubMed

Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

2013-01-01

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Altered tumor cell growth and tumorigenicity in models of microgravity

NASA Astrophysics Data System (ADS)

Yamauchi, K.; Taga, M.; Furian, L.; Odle, J.; Sundaresan, A.; Pellis, N.; Andrassy, R.; Kulkarni, A.

Spaceflight environment and microgravity (MG) causes immune dysfunction and is a major health risk to humans, especially during long-term space missions. The effects of microgravity environment on tumor growth and carcinogenesis are yet unknown. Hence, we investigated the effects of simulated MG (SMG) on tumor growth and tumorigenicity using in vivo and in vitro models. B16 melanoma cells were cultured in static flask (FL) and rotating wall vessel bioreactors (BIO) to measure growth and properties, melanin production and apoptosis. BIO cultures had 50% decreased growth (p<0.01), increased doubling time and a 150% increase in melanin production (p<0.05). Flow cytometric analysis showed increased apoptosis in BIO. When BIO cultured melanoma cells were inoculated sc in mice there was a significant increase in tumorigenicity as compared to FL cells. Thus SMG may have supported &selected highly tumorigenic cells and it is pos sible that in addition to decreased immune function MG may alter tumor cell characteristics and invasiveness. Thus it is important to study effects of microgravity environment and its stressors using experimental tumors and SMG to understand and evaluate carcinogenic responses to true microgravity. Further studies on carcinogenic events and their mechanisms will allow us develop and formulate countermeasures and protect space travelers. Additional results will be presented. (Supported by NASA NCC8-168 grant, ADK)

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development.

PubMed

Baxter, M A; Wynn, R F; Schyma, L; Holmes, D K; Wraith, J E; Fairbairn, L J; Bellantuono, I

2005-01-01

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

A novel perfused rotary bioreactor for cardiomyogenesis of embryonic stem cells.

PubMed

Teo, Ailing; Mantalaris, Athanasios; Song, Kedong; Lim, Mayasari

2014-05-01

Developments in bioprocessing technology play an important role for overcoming challenges in cardiac tissue engineering. To this end, our laboratory has developed a novel rotary perfused bioreactor for supporting three-dimensional cardiac tissue engineering. The dynamic culture environments provided by our novel perfused rotary bioreactor and/or the high-aspect rotating vessel produced constructs with higher viability and significantly higher cell numbers (up to 4 × 10(5) cells/bead) than static tissue culture flasks. Furthermore, cells in the perfused rotary bioreactor showed earlier gene expressions of cardiac troponin-T, α- and β-myosin heavy chains with higher percentages of cardiac troponin-I-positive cells and better uniformity of sacromeric α-actinin expression. A dynamic and perfused environment, as provided by this bioreactor, provides a superior culture performance in cardiac differentiation for embryonic stem cells particularly for larger 3D constructs.

Microenvironment Induced Spheroid to Sheeting Transition of Immortalized Human Keratinocytes (HaCaT) Cultured in Microbubbles Formed in Polydimethylsiloxane

PubMed Central

Chandrasekaran, Siddarth; Giang, Ut-Binh; King, Michael R.; DeLouise, Lisa A

2011-01-01

The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, and disease development and progression. In this study we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100–300 µm in diameter formed in hydrophobic polymer polydimethylsiloxane (PDMS) with ~60–100 µm circular openings and aspect ratio ~3.5. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15–20 cells / MB) and cultured under standard conditions, adopt a compact 3-D spheroidal morphology. Within 2–3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96 well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca2+ concentration and changes in Ecadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy. PMID:21724250

Detoxification of corn stover and corn starch pyrolysis liquors by Pseudomonas putida and Streptomyces setonii suspended cells and plastic compost support biofilms.

PubMed

Khiyami, Mohammad A; Pometto Iii, Anthony L; Brown, Robert C

2005-04-20

Plant biomass can be liquefied into fermentable sugars (levoglucosan then to glucose) for the production of ethanol, lactic acid, enzymes, and more by a process called pyrolysis. During the process microbial inhibitors are also generated. Pseudomonas putida (ATCC 17484) and Streptomyces setonii75Vi2 (ATCC 39116) were employed to degrade microbial inhibitors in diluted corn stover (Dcs) and diluted corn starch (Dst) pyrolysis liquors. The detoxification process evaluation included measuring total phenols and changes in UV spectra, a GC-MS analysis, and a bioassay, which employed Lactobacillus casei subsp. rhamosus (ATCC 11443) growth as an indicator of detoxification. Suspended-cell cultures illustrated limited detoxification ability of Dcs and Dst. P. putida and S. setoniiplastic compost support (PCS) biofilm continuous-stirred-tank-reactor pure cultures detoxified 10 and 25% (v/v) Dcs and Dst, whereas PCS biofilm mixed culture also partially detoxified 50% (v/v) Dcs and Dst in repeated batch culture. Therefore, PCS biofilm mixed culture is the process of choice to detoxify diluted pyrolysis liquors.

Development of a chick embryo heart cell for the cultivation of poliovirus.

PubMed

PRIER, J E; SULLIVAN, R

1959-04-17

An epithelial-like cell has been developed in line culture that apparently is stable. Although initially isolated cells were incapable of supporting the growth of poliovirus, the cells of the sixth and later passages allowed virus to propagate. The early, nonsusceptible cells were fibroblastic in appearance, in contrast to the epithelial type, poliovirussusceptible, derived cell of later passages.

Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI)

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. The beads are similar in size and density to human lymphoid cells. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light. In this photograph, beads are trapped in the injection port, with bubbles forming shortly after injection.

Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI)

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. The beads are similar in size and density to human lymphoid cells. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light. In this photograph, a TCM is shown after mixing protocols, and bubbles of various sizes can be seen.

High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

PubMed

Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

2012-07-01

In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

PubMed

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-05-04

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems

PubMed Central

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-01-01

Abstract Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations. PMID:29618087

Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix.

PubMed

Wang, Xuanzhi; Dai, Xingliang; Zhang, Xinzhi; Li, Xinda; Xu, Tao; Lan, Qing

2018-04-15

Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Hui; Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi; Pan, Wei-Kang

A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8more » was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin{sup +} cells decreased whilst the percentage of GFAP{sup +} cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.« less

Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

PubMed

Wang, D; Fløisand, Y; Myklebust, C V; Bürgler, S; Parente-Ribes, A; Hofgaard, P O; Bogen, B; Taskén, K; Tjønnfjord, G E; Schjesvold, F; Dalgaard, J; Tveita, A; Munthe, L A

2017-10-01

Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

Fifth Congress of Industrial Cell Technology 2014.

PubMed

Rasch, Anja

2015-01-01

The highly specialized and informative Fifth Congress of Industrial Cell Technology took place in Luebeck, close to Hamburg, on 11-12 September 2014. It was organized by the Fraunhofer Institution for Marine Biotechnology (EMB), Luebeck and supported by the cluster agency Life Science Nord Management GmbH as well as the Luebeck Chamber of Industry and Commerce. The central aim of the congress was to promote the name-giving platform applications of industrial cell technologies, in other words, the development of complex cell culture systems, analyzing technologies, innovative instruments and materials, etc. This year's sessions were: smart cell culture, bioreactor systems and cell goods including 3D bioprinting. This article highlights selected presentations of the congress.

Growth and development of cultured carrot cells and embryos under spaceflight conditions

NASA Technical Reports Server (NTRS)

Krikorian, A. D.; Dutcher, F. R.; Quinn, C. E.; Steward, F. C.

1981-01-01

Morphogenetically competent proembryonic cells and well-developed somatic embryos of carrot at two levels of organization were exposed for 18.5 days to a hypogravity environment aboard the Soviet Biosatellite Cosmos 1129. It was confirmed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots and minimally developed shoots. It was also shown that the space hypogravity environment could support the further growth of already-organized, later somatic embryonic stages and give rise to fully developed embryo-plantlets with roots and shoots.

Macrophage-selective toxicity as a mechanism of hydroquinone-induced myelotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, D.J.

1989-01-01

This research has focused upon the role of the bone marrow stroma in the etiology of benzene hematotoxicity. Treatment with the metabolite hydroquinone results in a reduced capacity of the stroma to support myelopoiesis. The goal of this research was to examine stromal cell selective toxicity following hydroquinone treatment. Populations of macrophages and a fibroblastoid cell line (LTF) or primary fibroblasts were developed from mouse bone marrow. Following treatment of with hydroquinone, treated or control fibroblastoid cells were reconstituted with control or treated macrophages, respectively, and the cultures were assayed for their ability to support myelopoiesis. To examine mechanisms ofmore » selective toxicity, macrophage and LTF cultures were incubated with 14C-hydroquinone and bioactivation was examined. After 24 hours, macrophages had 16-fold higher levels of bound {sup 14}C than LTF cells. Peroxide-dependent bioactivation in cell homogenates revealed that peroxide could support formation of covalent-binding species in macrophage homogenates but not in LTF homogenates. It was determined that macrophages, but not LTF cells, contained detectable levels of peroxidase activity which was consistent with the postulate that increased binding was due to peroxidase-mediated bioactivation of hydroquinone. Accordingly, purified myeloperoxidase incubated with {sup 14}C-hydroquinone, resulted in bioactivation to a covalent-binding species. This study provided evidence supporting selective bioactivation as a mechanism of selective toxicity of hydroquinone to bone marrow stromal macrophages.« less

Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

NASA Technical Reports Server (NTRS)

Shalhoub, V.; Conlon, D.; Tassinari, M.; Quinn, C.; Partridge, N.; Stein, G. S.; Lian, J. B.

1992-01-01

To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.

Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.

PubMed Central

Sutherland, H J; Lansdorp, P M; Henkelman, D H; Eaves, A C; Eaves, C J

1990-01-01

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells. Images PMID:2333304

Continuous Release of Tumor-Derived Factors Improves the Modeling of Cachexia in Muscle Cell Culture.

PubMed

Jackman, Robert W; Floro, Jess; Yoshimine, Rei; Zitin, Brian; Eiampikul, Maythita; El-Jack, Kahlid; Seto, Danielle N; Kandarian, Susan C

2017-01-01

Cachexia is strongly associated with a poor prognosis in cancer patients but the biological trigger is unknown and therefore no therapeutics exist. The loss of skeletal muscle is the most deleterious aspect of cachexia and it appears to depend on secretions from tumor cells. Models for studying wasting in cell culture consist of experiments where skeletal muscle cells are incubated with medium conditioned by tumor cells. This has led to candidates for cachectic factors but some of the features of cachexia in vivo are not yet well-modeled in cell culture experiments. Mouse myotube atrophy measured by myotube diameter in response to medium conditioned by mouse colon carcinoma cells (C26) is consistently less than what is seen in muscles of mice bearing C26 tumors with moderate to severe cachexia. One possible reason for this discrepancy is that in vivo the C26 tumor and skeletal muscle share a circulatory system exposing the muscle to tumor factors in a constant and increasing way. We have applied Transwell®-adapted cell culture conditions to more closely simulate conditions found in vivo where muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. C26 cells were incubated on a microporous membrane (a Transwell® insert) that constitutes the upper compartment of wells containing plated myotubes. In this model, myotubes are exposed to a constant supply of cancer cell secretions in the medium but without direct contact with the cancer cells, analogous to a shared circulation of muscle and cancer cells in tumor-bearing animals. The results for myotube diameter support the idea that the use of Transwell® inserts serves as a more physiological model of the muscle wasting associated with cancer cachexia than the bolus addition of cancer cell conditioned medium. The Transwell® model supports the notion that the dose and kinetics of cachectic factor delivery to muscle play a significant role in the extent of pathology.

Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.

PubMed

Lin, Youshan Melissa; Lim, Jessica Fang Yan; Lee, Jialing; Choolani, Mahesh; Chan, Jerry Kok Yen; Reuveny, Shaul; Oh, Steve Kah Weng

2016-06-01

Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

PubMed

He, Xiao-Tao; Wu, Rui-Xin; Xu, Xin-Yue; Wang, Jia; Yin, Yuan; Chen, Fa-Ming

2018-04-15

Accumulating evidence indicates that the physicochemical properties of biomaterials exert profound influences on stem cell fate decisions. However, matrix-based regulation selected through in vitro analyses based on a given cell population do not genuinely reflect the in vivo conditions, in which multiple cell types are involved and interact dynamically. This study constitutes the first investigation of how macrophages (Mφs) in stiffness-tunable transglutaminase cross-linked gelatin (TG-gel) affect the osteogenesis of bone marrow-derived mesenchymal stem cells (BMMSCs). When a single cell type was cultured, low-stiffness TG-gels promoted BMMSC proliferation, whereas high-stiffness TG-gels supported cell osteogenic differentiation. However, Mφs in high-stiffness TG-gels were more likely to polarize toward the pro-inflammatory M1 phenotype. Using either conditioned medium (CM)-based incubation or Transwell-based co-culture, we found that Mφs encapsulated in the low-stiffness matrix exerted a positive effect on the osteogenesis of co-cultured BMMSCs. Conversely, Mφs in high-stiffness TG-gels negatively affected cell osteogenic differentiation. When both cell types were cultured in the same TG-gel type and placed into the Transwell system, the stiffness-related influences of Mφs on BMMSCs were significantly altered; both the low- and high-stiffness matrix induced similar levels of BMMSC osteogenesis. Although the best material parameter for synergistically affecting Mφs and BMMSCs remains unknown, our data suggest that Mφ involvement in the co-culture system alters previously identified material-related influences on BMMSCs, such as matrix stiffness-related effects, which were identified based on a culture system involving a single cell type. Such Mφ-stem cell interactions should be considered when establishing proper matrix parameter-associated cell regulation in the development of biomimetic biomaterials for regenerative applications. The substrate stiffness of a scaffold plays critical roles in modulating both reparative cells, such as mesenchymal stem cells (MSCs), and immune cells, such as macrophages (Mφs). Although the influences of material stiffness on either Mφs or MSCs, have been extensively described, how the two cell types respond to matrix cues to dynamically affect each other in a three-dimensional (3D) biosystem remains largely unknown. Here, we report our findings that, in a platform wherein Mφs and bone marrow-derived MSCs coexist, matrix stiffness can influence stem cell fate through both direct matrix-associated regulation and indirect Mφ-based modulation. Our data support future studies of the MSC-Mφ-matrix interplay in the 3D context to optimize matrix parameters for the development of the next biomaterial. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Floating cultivation of marine cyanobacteria using coal fly ash.

PubMed

Matsumoto, M; Yoshida, E; Takeyama, H; Matsunaga, T

2000-01-01

The aim of this study was to develop improved methodologies for bulk culturing of biotechnologically useful marine cyanobacteria in the open ocean. We have investigated the viability of using coal fly ash (CFA) blocks as the support medium in a novel floating culture system for marine micro-algae. The marine cyanobacterium Synechococcus sp. NKBG 040607 was found to adhere to floating CFA blocks in liquid culture medium. Maximum density of attached cells of 2.0 x 10(8) cells/cm2 was achieved using seawater. The marine cyanobacterium Synechococcus sp. NKBG 042902 weakly adhered to floating CFA blocks in BG-11 medium. Increasing the concentration of calcium ion in the culture medium enhanced adherence to CFA blocks.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

PubMed Central

Sieh, Shirly; Taubenberger, Anna V.; Rizzi, Simone C.; Sadowski, Martin; Lehman, Melanie L.; Rockstroh, Anja; An, Jiyuan; Clements, Judith A.; Nelson, Colleen C.; Hutmacher, Dietmar W.

2012-01-01

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. PMID:22957009

Fanconi anemia mesenchymal stromal cells-derived glycerophospholipids skew hematopoietic stem cell differentiation through Toll-like receptor signaling

PubMed Central

Amarachintha, Surya; Sertorio, Mathieu; Wilson, Andrew; Li, Xiaoli; Pang, Qishen

2015-01-01

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids and their endogenous inhibitor, 5-(Tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells (HSPCs). We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (1) limiting-dilution CAFC assay revealed that TOFA significantly increased cobblestone colonies in Fanca−/− or Fancd2−/− co-cultures compared to untreated co-cultures. (2) Competitive repopulating assay using output cells collected from co-cultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca−/− or Fancd2−/− co-cultures. Further, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting Glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation. PMID:26212365

Biomimetic and synthetic esophageal tissue engineering.

PubMed

Jensen, Todd; Blanchette, Alex; Vadasz, Stephanie; Dave, Apeksha; Canfarotta, Michael; Sayej, Wael N; Finck, Christine

2015-07-01

A tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit. Donor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted. Isolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds. We demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

PubMed

Yan, Yuanwei; Song, Liqing; Tsai, Ang-Chen; Ma, Teng; Li, Yan

2016-01-01

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

Expansion of mesenchymal stem cells under atmospheric carbon dioxide.

PubMed

Brodsky, Arthur Nathan; Zhang, Jing; Visconti, Richard P; Harcum, Sarah W

2013-01-01

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system - which relies upon an elevated CO2 environment - typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO2 levels negatively affecting cell growth, but more importantly, the need to sparge CO2 into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO2 environment in static cultures. MSC expanded under atmospheric CO2 with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self-renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers.

Adaptation to Cadmium by Klebsiella aerogenes Growing in Continuous Culture Proceeds Mainly via Formation of Cadmium Sulfide

PubMed Central

Aiking, Harry; Kok, Karin; van Heerikhuizen, Harm; van 't Riet, Jan

1982-01-01

The adaptation of Klebsiella aerogenes to high levels of cadmium was studied in continuous culture under conditions of glucose limitation. When up to 6 × 10−4 M cadmium was added to a culture in steady state, growth ceased instantaneously but resumed within 5 h (dilution rate, 0.1 h−1). When again in steady state, these adapted cells exhibited a far greater tolerance to cadmium than did unadapted cells (not previously exposed to cadmium) when tested on solid media containing different concentrations of cadmium. This relative insensitivity of adapted cells to cadmium was subsequently lost in continuous culture within 5 days after omitting cadmium from the influent medium. Thus, the phenomenon was an inducible physiological process. Adapted cells contained substantial amounts of cadmium (up to 2.4% of the bacterial dry weight). The cadmium content of the cells was dependent on growth conditions and was found to be proportional to the inorganic sulfide content of the cells in all cases. This suggested that formation of CdS is probably the most important mechanism of detoxification in this organism. The presence of large numbers of electron-dense granules on the cell surface (absent in cultures without added cadmium) provided additional support for this conclusion. Images PMID:16346119

Three-dimensional co-culture process

NASA Technical Reports Server (NTRS)

Wolf, David A. (Inventor); Goodwin, Thomas J. (Inventor)

1992-01-01

The present invention relates to a 3-dimensional co-culture process, more particularly to methods or co-culturing at least two types of cells in a culture environment, either in space or in unit gravity, with minimum shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region to form 3-dimensional tissue-like structures. Several examples of multicellular 3-dimensional experiences are included. The protocol and procedure are also set forth. The process allows simultaneous culture of multiple cell types and supporting substrates in a manner which does not disrupt the 3-dimensional spatial orientation of these components. The co-cultured cells cause a mutual induction effect which mimics the natural hormonal signals and cell interactions found in the intact organism. This causes the tissues to differentiate and form higher 3-dimensional structures such as glands, junctional complexes polypoid geometries, and microvilli which represent the corresponding in-vitro structures to a greater degree than when the cell types are cultured individually or by conventional processes. This process was clearly demonstrated for the case of two epithelial derived colon cancer lines, each co-cultured with normal human fibroblasts and with microcarrier bead substrates. The results clearly demonstrate increased 3-dimensional tissue-like structure and biochemical evidence of an increased differentiation state. With the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and has some of the characteristics of in-vitro tissue. The process provides enhanced 3-dimensional tissue which create a multicellular organoid differentiation model.

Serum replacement with a growth factor-free synthetic substance in culture medium contributes to effective establishment of mouse embryonic stem cells of various origins.

PubMed

Lee, Seung Tae; Oh, Se Woong; Kim, Dae Yong; Han, Jae Yong; Moon, Shin Yong; Lim, Jeong Mook

2006-10-01

To evaluate whether serum replacement with growth factor-free synthetic substances contributed to the effective establishment of embryonic stem (ES) cells. Randomized, prospective model study. Gamete and stem cell biotechnology laboratory at Seoul National University in Korea. F1 (C57BL6 x DBA2) mice. Blastocysts of different origins were cultured in serum-replaced media. Embryonic stem cell establishment. Eight batches of ES cells were established from colony-forming inner cell mass cells after the replacement of fetal bovine serum (FBS) with synthetic knockout serum replacement (KSR) in mkDMEM. The established cells were positive for ES cell markers and formed both embryoid bodies in vitro and teratomas in vivo, but the established cell batches and control (transformed) ES cells responded differently to the culture media. Higher levels of cell viability were detected after the replacement with the 75:25 FBS-KSR mixture than with any other mixtures, and a gradual decrease in viability was detected as the KSR volume ratio was increased. The 75:25 FBS-KSR mixture-containing medium supported ES cell establishment of outbred ICR, F1, and F2 of C57BL6/DBA2; F1 parthenogenetic and ES cell-complemented tetraploid blastocysts; and single ES-cell cultures. A serum-replaced medium could be used for effective ES-cell establishment of various origins.

Mature Hepatocytes Exhibit Unexpected Plasticity by Direct Dedifferentiation into Liver Progenitor Cells in Culture

PubMed Central

Chen, Yixin; Wong, Philip P.; Sjeklocha, Lucas; Steer, Clifford J.; Sahin, M. Behnan

2011-01-01

Although there have been numerous reports describing the isolation of liver progenitor cells from adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly-purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, Liver Derived Progenitor Cells or LDPCS, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within one week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in generation of β-galactosidase-positive liver progenitor cells demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy. Conclusion Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture; and this may potentially have a significant impact on the treatment of liver diseases requiring liver or hepatocyte transplantation. PMID:21953633

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram

2013-11-20

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitivemore » kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.« less

The PCC assay can be used to predict radiosensitivity in biopsy cultures irradiated with different types of radiation.

PubMed

Suzuki, Masao; Tsuruoka, Chizuru; Nakano, Takashi; Ohno, Tatsuya; Furusawa, Yoshiya; Okayasu, Ryuichi

2006-12-01

The aim of this study was to identify potential biomarkers for radiosensitivity using the relationship between cell killing and the yield of excess chromatin fragments detected with the premature chromosome condensation (PCC) technique. This method was applied to primary cultured cells obtained from biopsies from patients. Six primary culture biopsies were obtained from 6 patients with carcinoma of the cervix before starting radiotherapy. The cultures were irradiated with two different LET carbon-ion beams (LET = 13 keV/microm, 77.1+/-2.8 keV/microm) and 200 kV X-rays. The carbon-ion beams were produced by Heavy Ion Medical Accelerator in Chiba (HIMAC). PCC was performed using the polyethylene glycol-mediated cell fusion technique. The yield of excess chromatin fragments were measured by counting the number of unrejoined chromatin fragments detected with the PCC technique after a 24-h post-irradiation incubation period. Obtained results indicated that cultures which were more sensitive to killing were also more susceptible to the induction of excess chromatin fragments. Furthermore there was a good correlation between cell killing and excess chromatin fragments among the 6 cell cultures examined. There is also evidence that the induction of excess chromatin fragments increased with increasing LET as well as cell-killing effect in the same cell culture. The data reported here support the idea that the yield of excess chromatin fragments detected with the PCC technique might be useful for predicting the radiosensitivity of cells contained in tumor tissue, and to predict responses to different radiation types.

Ex-vivo expansion of hematopoietic progenitor cells: preliminary results in breast cancer.

PubMed

McNiece, I; Jones, R; Cagnoni, P; Bearman, S; Nieto, Y; Shpall, E J

1999-04-01

Ex-vivo expanded progenitor cells have been proposed as a source of cells to support high-dose chemotherapy and to decrease or eliminate the period of neutropenia following transplantation. To date, no clinical studies using ex vivo expanded cells, have demonstrated any decrease in the time to neutrophil or platelet recovery, although a number of clinical studies have been performed using a variety of growth factor cocktails and culture conditions. Over the past 6 years we have developed a static culture system that results in optimal expansion of myeloid progenitor cells. We have initiated a clinical study to evaluate this culture system in breast cancer patients receiving peripheral blood progenitor cells (PBPC) to support high-dose chemotherapy. CD34 selected cells were cultured for 10 days in 800 ml of defined media (Amgen Inc.) containing 100 ng/ml each of rhSCF, rhG-CSF and rhMGDF in 1L teflon bags (American Fluoroseal) at 20,000 to 50,000 cells per ml. After culture the cells were washed with 3 volumes of PBS to remove all media and growth factors and reinfused on day 0 of transplant followed by daily administration of rhG-CSF. On day +1 the patients received an unexpanded PBPC product to ensure the durability of the graft. Patients transplanted with expanded PBPC cells recovered neutrophil counts (ANC > 500/microl) as early as day 4 post transplant with a median of 6 days (range 4 to 14 days). In comparison, our historical control group of patients (N=175) had a median time to neutrophil engraftment of 9 days (range 7 to 24 days). A second cohort of patients were transplanted with expanded cells alone and a similar rapid engraftment was obtained. The first patients are now over 70 days post transplant with durable engraftment. No effect on platelet recovery has been observed in any patients to date. These data demonstrate that PBPC expanded under the conditions defined can significantly shorten the time to engraftment of neutrophils.

Culturability as an indicator of succession in microbial communities

NASA Technical Reports Server (NTRS)

Garland, J. L.; Cook, K. L.; Adams, J. L.; Kerkhof, L.

2001-01-01

Successional theory predicts that opportunistic species with high investment of energy in reproduction and wide niche width will be replaced by equilibrium species with relatively higher investment of energy in maintenance and narrower niche width as communities develop. Since the ability to rapidly grow into a detectable colony on nonselective agar medium could be considered as characteristic of opportunistic types of bacteria, the percentage of culturable cells may be an indicator of successional state in microbial communities. The ratios of culturable cells (colony forming units on R2A agar) to total cells (acridine orange direct microscopic counts) and culturable cells to active cells (reduction of 5-cyano-2,3-ditolyl tetrazolium chloride) were measured over time in two types of laboratory microcosms (the rhizosphere of hydroponically grown wheat and aerobic, continuously stirred tank reactors containing plant biomass) to determine the effectiveness of culturabilty as an index of successional state. The culturable cell:total cell ratio in the rhizosphere decreased from approximately 0.25 to less than 0.05 during the first 30-50 days of plant growth, and from 0.65 to 0.14 during the first 7 days of operation of the bioreactor. The culturable cell:active cell ratio followed similar trends, but the values were consistently greater than the culturable cell:total cell ratio, and even exceeded I in early samples. Follow-up studies used a cultivation-independent method, terminal restriction fragment length polymorphisms (TRFLP) from whole community DNA, to assess community structure. The number of TRFLP peaks increased with time, while the number of culturable types did not, indicating that the general decrease in culturability is associated with a shift in community structure. The ratio of respired to assimilated C-14-labeled amino acids increased with the age of rhizosphere communities, supporting the hypothesis that a shift in resource allocation from growth to maintenance occurs with time. Results from this work indicate that the percentage of culturable cells may be a useful method for assessing the successional state of microbial communities.

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

PubMed

Ishihara, Keisuke; Ranga, Adrian; Lutolf, Matthias P; Tanaka, Elly M; Meinhardt, Andrea

2017-01-01

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds.

PubMed

Mohanty, Soumyaranjan; Larsen, Layla Bashir; Trifol, Jon; Szabo, Peter; Burri, Harsha Vardhan Reddy; Canali, Chiara; Dufva, Marin; Emnéus, Jenny; Wolff, Anders

2015-10-01

One of the major challenges in producing large scale engineered tissue is the lack of ability to create large highly perfused scaffolds in which cells can grow at a high cell density and viability. Here, we explore 3D printed polyvinyl alcohol (PVA) as a sacrificial mould in a polymer casting process. The PVA mould network defines the channels and is dissolved after curing the polymer casted around it. The printing parameters determined the PVA filament density in the sacrificial structure and this density resulted in different stiffness of the corresponding elastomer replica. It was possible to achieve 80% porosity corresponding to about 150 cm(2)/cm(3) surface to volume ratio. The process is easily scalable as demonstrated by fabricating a 75 cm(3) scaffold with about 16,000 interconnected channels (about 1m(2) surface area) and with a channel to channel distance of only 78 μm. To our knowledge this is the largest scaffold ever to be produced with such small feature sizes and with so many structured channels. The fabricated scaffolds were applied for in-vitro culturing of hepatocytes over a 12-day culture period. Smaller scaffolds (6×4 mm) were tested for cell culturing and could support homogeneous cell growth throughout the scaffold. Presumably, the diffusion of oxygen and nutrient throughout the channel network is rapid enough to support cell growth. In conclusion, the described process is scalable, compatible with cell culture, rapid, and inexpensive. Copyright © 2015. Published by Elsevier B.V.

Lindane removal by pure and mixed cultures of immobilized actinobacteria.

PubMed

Saez, Juliana M; Benimeli, Claudia S; Amoroso, María J

2012-11-01

Lindane (γ-HCH) is an organochlorine insecticide that has been widely used in developing countries. It is known to persist in the environment and can cause serious health problems. One of the strategies adopted to remove lindane from the environment is bioremediation using microorganisms. Immobilized cells present advantages over free suspended cells, like their high degradation efficiency and protection against toxins. The aims of this work were: (1) To evaluate the ability of Streptomyces strains immobilized in four different matrices to remove lindane, (2) To select the support with optimum lindane removal by pure cultures, (3) To assay the selected support with consortia and (4) To evaluate the reusability of the immobilized cells. Four Streptomyces sp. strains had previously shown their ability to grow in the presence of lindane. Lindane removal by microorganisms immobilized was significantly higher than in free cells. Specifically immobilized cells in cloth sachets showed an improvement of around 25% in lindane removal compared to the abiotic control. Three strains showed significantly higher microbial growth when they were entrapped in silicone tubes. Strains immobilized in PVA-alginate demonstrated lowest growth. Mixed cultures immobilized inside cloth sachets showed no significant enhancement compared to pure cultures, reaching a maximum removal of 81% after 96 h for consortium I, consisting of the four immobilized strains together. Nevertheless, the cells could be reused for two additional cycles of 96 h each, obtaining a maximum removal efficiency of 71.5% when each of the four strains was immobilized in a separate bag (consortium III). Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro effects of simulated microgravity on Sertoli cell function

NASA Astrophysics Data System (ADS)

Masini, M. A.; Prato, P.; Scarabelli, L.; Lanza, C.; Palmero, S.; Pointis, G.; Ricci, F.; Strollo, F.

2011-02-01

With the advent of space flights questions concerning the effects of microgravity (0×G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0×G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1×G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0×G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1×G samples. Initially, 0×G seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0×G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.

Behavior of a Spontaneously Arising Human Retinal Pigment Epithelial Cell Line Cultivated on Thin Alginate Film.

PubMed

Najafabadi, Hoda Shams; Soheili, Zahra-Soheila; Ganji, Shahla Mohammad

2015-01-01

A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide. Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle's-medium-and-Ham's-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks. The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features. Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures.

Behavior of a Spontaneously Arising Human Retinal Pigment Epithelial Cell Line Cultivated on Thin Alginate Film

PubMed Central

Najafabadi, Hoda Shams; Soheili, Zahra-Soheila; Ganji, Shahla Mohammad

2015-01-01

Purpose: A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide. Methods: Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle’s-medium-and-Ham’s-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks. Results: The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features. Conclusion: Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures. PMID:26730315

Enhanced production of human influenza virus in PBS-12SF cells with a reduced interferon response.

PubMed

Carvajal-Yepes, Monica; Sporer, Kelly R B; Carter, Jenna L; Colvin, Christopher J; Coussens, Paul M

2015-01-01

Influenza is one of the most important infectious diseases in humans. The best way to prevent severe illness caused by influenza infection is vaccination. Cell culture-derived influenza vaccines are being considered in addition to the widely used egg-based system in order to support the increasing seasonal demand and to be prepared in case of a pandemic. Cell culture based systems offer increased safety, capacity, and flexibility with reduced downstream processing relative to embryonated eggs. We have previously reported a chick embryo cell line, termed PBS-12SF, that supports replication of human and avian influenza A viruses to high titers (>10(7) PFU/ml) without the need for exogenous proteases or serum proteins. Viral infections in cells are limited by the Interferon (IFN) response typified by production of type I IFNs that bind to the IFNα/β receptor and activate an antiviral state. In this study, we investigated how neutralizing the interferon (IFN) response in PBS-12SF cells, via shRNA-mediated knock-down of IFNAR1 mRNA expression, affects influenza virus production. We were successful in knocking down ∼90% of IFNAR1 protein expression by this method, resulting in a significant decrease in the response to recombinant chIFNα stimulation in PBS-12SF cells as shown by a reduction in expression of interferon-responsive genes when compared to control cells. Additionally; IFNAR1-knock-down cells displayed enhanced viral HA production and released more virus into cell culture supernatants than parental PBS-12SF cells.

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

PubMed Central

Gardner, Jameson K.; Herbst-Kralovetz, Melissa M.

2016-01-01

The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies. PMID:27834891

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

PubMed Central

Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions.

PubMed

Patrikoski, Mimmi; Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

Megakaryocyte Polyploidization and Proplatelet Formation in Low-Attachment Conditions.

PubMed

Schlinker, Alaina C; Duncan, Mark T; DeLuca, Teresa A; Whitehead, David C; Miller, William M

2016-07-15

In vitro -derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture.

Manipulation of immune system via immortal bone marrow stem cells.

PubMed

Ruedl, Christiane; Khameneh, Hanif Javanmard; Karjalainen, Klaus

2008-09-01

Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98-HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.

A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity.

PubMed

Sherman, Hilary; Gitschier, Hannah J; Rossi, Ann E

2018-01-01

The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo -like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1α). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo -like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

Comprehensive proteomic characterization of stem cell-derived extracellular matrices.

PubMed

Ragelle, Héloïse; Naba, Alexandra; Larson, Benjamin L; Zhou, Fangheng; Prijić, Miralem; Whittaker, Charles A; Del Rosario, Amanda; Langer, Robert; Hynes, Richard O; Anderson, Daniel G

2017-06-01

In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles1

PubMed Central

Xu, Min; West-Farrell, Erin R.; Stouffer, Richard L.; Shea, Lonnie D.; Woodruff, Teresa K.; Zelinski, Mary B.

2009-01-01

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for ≤30 days. Follicles isolated from the follicular phase of the menstrual cycle had a higher survival rate (P < 0.05) than those isolated from the luteal phase; however, this difference may also be attributed to differing sizes of follicles isolated during the different stages. Follicles survived and grew in two hydrogel conditions (0.5% and 0.25% alginate). Follicle diameters increased to a greater extent (P < 0.05) in the presence of FSH alone than in FSH plus LH. Regardless of gonadotropin treatment, follicles produced estradiol, androstenedione, and progesterone by 14–30 days in vitro. Thus, an alginate hydrogel maintains the 3-D structure of individual secondary macaque follicles, permits follicle growth, and supports steroidogenesis for ≤30 days in vitro. This study documents the first use of the alginate system to maintain primate tissue architecture, and findings suggest that encapsulated 3-D culture will be successful in supporting the in vitro development of human follicles. PMID:19474063

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

PubMed

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Conducting polymer scaffolds for electrical control of cellular functions (Conference Presentation)

NASA Astrophysics Data System (ADS)

Inal, Sahika; Wan, Alwin M.; Williams, Tiffany V.; Giannelis, Emmanuel P.; Fischbach-Teschl, Claudia; Gourdon, Delphine; Owens, Róisín. M.; Malliaras, George G.

2016-09-01

Considering the limited physiological relevance of 2D cell culture experiments, significant effort was devoted to the development of materials that could more accurately recreate the in vivo cellular microenvironment, and support 3D cell cultures in vitro. (1) One such class of materials is conducting polymers, which are promising due to their compliant mechanical properties, compatibility with biological systems, mixed electrical and ionic conductivity, and ability to form porous structures. (2) In this work, we report the fabrication of a single component, macroporous scaffold made from poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) via an ice-templating method. (3) PEDOT:PSS scaffolds offer tunable pore size, morphology and shape through facile changes in preparation conditions, and are capable of supporting 3D cell cultures due to their biocompatibility and tissue-like elasticity. Moreover, these materials are functional: they exhibit excellent electrochemical switching behavior and significantly lower impedance compared to films. Their electrochemical activity enables their use in the active channel of a state of the art diagnostic tool in the field of bioelectronics, i.e., the organic electrochemical transistor (OECT). The inclusion of cells within the porous architecture affects the impedance of the electrically-conducting polymer network and, thus, may be used as a method to quantify cell growth. The adhesion and pro-angiogenic secretions of mouse fibroblasts cultured within the scaffolds can be controlled by switching the electrochemical state of the polymer prior to cell-seeding. In summary, these smart materials hold promise not only as extracellular matrix-mimicking structures for cell culture, but also as high-performance bioelectronic tools for diagnostic and signaling applications. References [1] M. Holzwarth, P. X. Ma, Journal of Materials Chemistry, 21, 10243-10251 (2011). [2] L. H. Jimison, J. Rivnay, R. M. Owens, in Organic Electronics, Wiley-VCH Verlag GmbH and Co. KGaA, 27-6 (2013). [3] A. M.-D. Wan, S. Inal, T. Williams et al. Journal of Materials Chemistry B, DOI: 10.1039/C5TB00390C (2015).

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks.

PubMed

Suarez-Martinez, Ariana D; Bierschenk, Susanne; Huang, Katie; Kaplan, Dana; Bayer, Carolyn L; Meadows, Stryder M; Sperandio, Markus; Murfee, Walter L

2018-05-18

The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study's objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks. © 2018 S. Karger AG, Basel.

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

PubMed

Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra

2017-11-01

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Polyisocyanopeptide hydrogels: A novel thermo-responsive hydrogel supporting pre-vascularization and the development of organotypic structures.

PubMed

Zimoch, Jakub; Padial, Joan Simó; Klar, Agnes S; Vallmajo-Martin, Queralt; Meuli, Martin; Biedermann, Thomas; Wilson, Christopher J; Rowan, Alan; Reichmann, Ernst

2018-04-01

Molecular and mechanical interactions with the 3D extracellular matrix are essential for cell functions such as survival, proliferation, migration, and differentiation. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. This is a synthetic, thermo-responsive and stress-stiffening material synthesized via polymerization of the corresponding monomers using a nickel perchlorate as a catalyst. It can be tailored to meet various demands of cells by modulating its stiffness and through the decoration of the polymer with short GRGDS peptides using copper free click chemistry. These peptides make the hydrogels biocompatible by mimicking the binding sites of certain integrins. This study focuses on the optimization of the PIC polymer properties for efficient cell, tissue and organ development. Screening for the optimal stiffness of the hydrogel and the ideal concentration of the GRGDS ligand conjugated with the polymer, enabled cell proliferation, migration and differentiation of various primary cell types of human origin. We demonstrate that fibroblasts, endothelial cells, adipose-derived stem cells and melanoma cells, do survive, thrive and differentiate in optimized PIC hydrogels. Importantly, these hydrogels support the spontaneous formation of complex structures like blood capillaries in vitro. Additionally, we utilized the thermo-responsive properties of the hydrogels for a rapid and gentle recovery of viable cells. Finally, we show that organotypic structures of human origin grown in PIC hydrogels can be successfully transplanted subcutaneously onto immune-compromised rats, on which they survive and integrate into the surrounding tissue. Molecular and mechanical interactions with the surrounding environment are essential for cell functions. Although 2D culture systems greatly contributed to our understanding of complex biological phenomena, they cannot substitute for crucial interaction that take place in 3D. 3D culture systems aim to overcome limitations of the 2D cultures and answer new questions about cell functions. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. They are synthetic and can be tailor to meet certain experimental demands. Additionally, they are characterized by strain-stiffening, a feature crucial for cell behaviour, but rare in hydrogels. Their thermos-responsive properties enable quick recovery of the cells by a simple procedure of lowering the temperature. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

PubMed

Kaneko, Ai; Sankai, Yoshiyuki

2014-01-01

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

PubMed

Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

2011-08-01

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

PubMed Central

Nasu, Makoto; Takata, Nozomu; Danjo, Teruko; Sakaguchi, Hideya; Kadoshima, Taisuke; Futaki, Sugiko; Sekiguchi, Kiyotoshi; Eiraku, Mototsugu; Sasai, Yoshiki

2012-01-01

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development. PMID:23300850

Formalin produces depolarizations in human airway smooth muscle in vitro.

PubMed

Richards, Ira S; DeHate, Robin B

2006-03-01

Respiratory irritants may result in airway smooth muscle (ASM) depolarization and bronchoconstriction. We examined the effect of formalin on membrane potentials in human ASM in two types of in vitro preparations: strip preparations, which contain functional sensory and motor nerve endings and cultured cells, which lack these nerve endings due to the tissue dissociation process. Depolarizations occurred in atropine-treated strip preparations in response to formalin exposures, but not in similarly-treated cultured cells, suggesting a role for non-cholinergic mediators in formalin-induced depolarization. It is suggested that formalin may act as an irritant to produce bronchoconstriction that is mediated by the release of endogenous substance P (SP) from peripheral sensory nerve endings. This is supported by our observation that exogenous SP produced depolarizations of a magnitude similar to those produced by formalin in both strip preparations and cultured cells. In addition, capsaicin, which releases endogenous SP from nerve endings, produced depolarizations of a magnitude similar to formalin in strip preparations, but was without effect in cultured cells.

Expansion and Differentiation of Germline-Derived Pluripotent Stem Cells on Biomaterials

PubMed Central

Šarić, Tomo; Denecke, Bernd; Peinkofer, Gabriel; Bovi, Manfred; Groll, Jürgen; Ko, Kinarm; Salber, Jochen; Halbach, Marcel; Schöler, Hans R.; Zenke, Martin; Neuss, Sabine

2013-01-01

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer® LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level. PMID:23234562

Expansion and differentiation of germline-derived pluripotent stem cells on biomaterials.

PubMed

Hoss, Mareike; Šarić, Tomo; Denecke, Bernd; Peinkofer, Gabriel; Bovi, Manfred; Groll, Jürgen; Ko, Kinarm; Salber, Jochen; Halbach, Marcel; Schöler, Hans R; Zenke, Martin; Neuss, Sabine

2013-05-01

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer(®) LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level.

Polyurethane/polylactide-based biomaterials combined with rat olfactory bulb-derived glial cells and adipose-derived mesenchymal stromal cells for neural regenerative medicine applications.

PubMed

Grzesiak, Jakub; Marycz, Krzysztof; Szarek, Dariusz; Bednarz, Paulina; Laska, Jadwiga

2015-01-01

Research concerning the elaboration and application of biomaterial which may support the nerve tissue regeneration is currently one of the most promising directions. Biocompatible polymer devices are noteworthy group among the numerous types of potentially attractive biomaterials for regenerative medicine application. Polylactides and polyurethanes may be utilized for developing devices for supporting the nerve regeneration, like nerve guide conduits or bridges connecting the endings of broken nerve tracts. Moreover, the combination of these biomaterial devices with regenerative cell populations, like stem or precursor cells should significantly improve the final therapeutic effect. Therefore, the composition and structure of final device should support the proper adhesion and growth of cells destined for clinical application. In current research, the three polymer mats elaborated for connecting the broken nerve tracts, made from polylactide, polyurethane and their blend were evaluated both for physical properties and in vitro, using the olfactory-bulb glial cells and mesenchymal stem cells. The evaluation of Young's modulus, wettability and roughness of obtained materials showed the differences between analyzed samples. The analysis of cell adhesion, proliferation and morphology showed that the polyurethane-polylactide blend was the most neutral for cells in culture, while in the pure polymer samples there were significant alterations observed. Our results indicated that polyurethane-polylactide blend is an optimal composition for culturing and delivery of glial and mesenchymal stem cells. Copyright © 2015. Published by Elsevier B.V.

Evaluation of the potential of novel PCL-PPDX biodegradable scaffolds as support materials for cartilage tissue engineering.

PubMed

Chaim, Isaac A; Sabino, Marcos A; Mendt, Mayela; Müller, Alejandro J; Ajami, Diana

2012-04-01

Cartilage is a specialized tissue represented by a group of particular cells (the chondrocytes) and an abundant extracellular matrix. Because of the reduced regenerative capacity of this tissue, cartilage injuries are often difficult to handle. Nowadays tissue engineering has emerged as a very promising discipline, and biodegradable polymeric scaffolds are widely used as tissue supports. In cartilage injuries, the use of autologous chondrocyte implantation from non-affected cartilage zones has emerged as a very interesting technique, where chondrocytes are expanded in order to obtain a greater number of cells. Nevertheless, it has been reported that chondrocytes in bidimensional cultures suffer a dedifferentiation process. The present study sought, in the first place, to standardize a novel protocol in order to obtain primary cultures of chondrocytes from newborn rabbit hyaline cartilage from the xiphoid process. Second, the potential of porous three-dimensional (3D) biodegradable polymeric matrices as support materials for chondrocytes was evaluated: a novel poly(ε-caprolactone)-poly(p-dioxanone) (PCL-PPDX) blend in a 90:10 w:w ratio and poly(ε-caprolactone) (PCL). After achieving the standardization, a typical round-shaped chondrocyte morphology and the expression of collagen type II and aggrecan, evaluated by RT-PCR, were observed. Second-passage chondrocytes adhered effectively to these scaffolds, although cell growth at 7 days in culture was significantly less in the PCL-PPDX blend. After 3 weeks of culture on PCL-PPDX or PCL, the cells expressed collagen type II. The present study demonstrates the potential, unknown until now, of PCL-PPDX blend scaffolds in the field of cartilage tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd.

CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro

PubMed Central

Hansen, Hinrich P.; Trad, Ahmad; Dams, Maria; Zigrino, Paola; Moss, Marcia; Tator, Maximilian; Schön, Gisela; Grenzi, Patricia C; Bachurski, Daniel; Aquino, Bruno; Dürkop, Horst; Reiners, Katrin S; von Bergwelt-Baildon, Michael; Hallek, Michael; Grötzinger, Joachim; Engert, Andreas; Leme, Adriana F Paes; von Strandmann, Elke Pogge

2016-01-01

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30−/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs. PMID:27105521

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation.

PubMed

Akbarian, Vahe; Wang, Weijia; Audet, Julie

2012-05-01

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture. Copyright © 2012 International Society for Advancement of Cytometry.

Culture Supernatants of Lactobacillus gasseri and L. crispatus Inhibit Candida albicans Biofilm Formation and Adhesion to HeLa Cells.

PubMed

Matsuda, Yuko; Cho, Otomi; Sugita, Takashi; Ogishima, Daiki; Takeda, Satoru

2018-03-30

Vulvovaginal candidiasis (VVC) is a common superficial infection of the vaginal mucous membranes caused by the fungus Candida albicans. The aim of this study was to assess the mechanisms underlying the inhibitory effects of the culture supernatants of Lactobacillus gasseri and L. crispatus, the predominant microbiota in Asian healthy women, on C. albicans biofilm formation. The inhibition of C. albicans adhesion to HeLa cells by Lactobacillus culture supernatant was also investigated. Candida albicans biofilm was formed on polystyrene flat-bottomed 96-well plates, and the inhibitory effects on the initial colonization and maturation phases were determined using the XTT reduction assay. The expression levels of biofilm formation-associated genes (HWP1, ECE1, ALS3, BCR1, EFG1, TEC1, and CPH1) were determined by reverse transcription quantitative polymerase chain reaction. The inhibition of C. albicans adhesion to HeLa cells by Lactobacillus culture supernatant was evaluated by enumerating viable C. albicans cells. The culture supernatants of both Lactobacillus species inhibited the initial colonization and maturation of C. albicans biofilm. The expression levels of all biofilm formation-related genes were downregulated in the presence of Lactobacillus culture supernatant. The culture supernatant also inhibited C. albicans adhesion to HeLa cells. The culture supernatants of L. gasseri and L. crispatus inhibited C. albicans biofilm formation by downregulating biofilm formation-related genes and C. albicans adhesion to HeLa cells. These findings support the notion that Lactobacillus metabolites may be useful alternatives to antifungal drugs for the management of VVC.

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media.

PubMed

Meunier, Sarah M; Todorovic, Biljana; Dare, Emma V; Begum, Afroza; Guillemette, Simon; Wenger, Andrew; Saxena, Priyanka; Campbell, J Larry; Sasges, Michael; Aucoin, Marc G

2016-01-01

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.

Aneuploidy in immortalized human mesenchymal stem cells with non-random loss of chromosome 13 in culture.

PubMed

Takeuchi, Masao; Takeuchi, Kikuko; Ozawa, Yutaka; Kohara, Akihiro; Mizusawa, Hiroshi

2009-01-01

Aneuploidy (an abnormal number of chromosomes) is commonly observed in most human cancer cells, highlighting the need to examine chromosomal instability in tumorigenesis. Previously, the immortalized human mesenchymal stem cell line UE6E7T-3 was shown to undergo a preferential loss of one copy of chromosome 13 after prolonged culture. Here, the loss of chromosome 13 was found to be caused by chromosome missegregation during mitosis, which involved unequal segregation, exclusion of the misaligned chromosome 13 on the metaphase plate, and trapping of chromosome 13 in the midbody region, as observed by fluorescence in situ hybridization. Near-diploid aneuploidy, not tetraploidy, was the direct result. The loss of chromosome 13 was non-random, and was detected by analysis of microsatellites and single nucleotide polymorphism-based loss of heterozygosity (LOH). Of the five microsatellite loci on chromosome 13, four loci showed microsatellite instability at an early stage in culture, and LOH was apparent at a late stage in culture. These results suggest that the microsatellite mutations cause changes in centromere integrity provoking loss of this chromosome in the UE6E7T-3 cell line. Thus, these results support the use of this cell line as a useful model for understanding the mechanism of aneuploid formation in cell cultures.

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media

PubMed Central

Meunier, Sarah M.; Todorovic, Biljana; Dare, Emma V.; Begum, Afroza; Guillemette, Simon; Wenger, Andrew; Saxena, Priyanka; Campbell, J. Larry; Sasges, Michael; Aucoin, Marc G.

2016-01-01

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media. PMID:26975046

Synthetic PEG Hydrogel for Engineering the Environment of Ovarian Follicles.

PubMed

Mendez, Uziel; Zhou, Hong; Shikanov, Ariella

2018-01-01

The functional unit within the ovary is the ovarian follicle, which is also a morphological unit composed of three basic cell types: the oocyte, granulosa, and theca cells. Similar to human ovarian follicles, mouse follicles can be isolated from their ovarian environment and cultured in vitro to study folliculogenesis, or follicle development for days or weeks. Over the course of the last decade, follicle culture in a three-dimensional (3D) environment exponentially improved the outcomes of in vitro folliculogenesis. Follicle culture in 3D environments preserves follicle architecture and promotes the cross talk between cells in the follicle. Hydrogels, such as polyethylene glycol (PEG), have been used for various physiological systems for regenerative purposes because they provide a 3D environment similar to soft tissues, allow diffusion of nutrients, and can be readily modified to present biological signals, including cell adhesion ligands and proteolytic degradation facilitated by enzymes secreted by the encapsulated cells. This chapter outlines the application of PEG hydrogels to the follicle culture, including the procedures to isolate, encapsulate, and culture mouse ovarian follicles. The tunable properties of PEG hydrogels support co-encapsulation of ovarian follicles with somatic cells, which further promote follicle survival and growth in vitro through paracrine and juxtacrine interactions.

The Biotechnology Facility for International Space Station

NASA Technical Reports Server (NTRS)

Goodwin, Thomas; Lundquist, Charles; Hurlbert, Katy; Tuxhorn, Jennifer

2004-01-01

The primary mission of the Cellular Biotechnology Program is to advance microgravity as a tool in basic and applied cell biology. The microgravity environment can be used to study fundamental principles of cell biology and to achieve specific applications such as tissue engineering. The Biotechnology Facility (BTF) will provide a state-of-the-art facility to perform cellular biotechnology research onboard the International Space Station (ISS). The BTF will support continuous operation, which will allow performance of long-duration experiments and will significantly increase the on-orbit science throughput. With the BTF, dedicated ground support, and a community of investigators, the goals of the Cellular Biotechnology Program at Johnson Space Center are to: Support approximately 400 typical investigator experiments during the nominal design life of BTF (10 years). Support a steady increase in investigations per year, starting with stationary bioreactor experiments and adding rotating bioreactor experiments at a later date. Support at least 80% of all new cellular biotechnology investigations selected through the NASA Research Announcement (NRA) process. Modular components - to allow sequential and continuous experiment operations without cross-contamination Increased cold storage capability (+4 C, -80 C, -180 C). Storage of frozen cell culture inoculum - to allow sequential investigations. Storage of post-experiment samples - for return of high quality samples. Increased number of cell cultures per investigation, with replicates - to provide sufficient number of samples for data analysis and publication of results in peer-reviewed scientific journals.

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival.

PubMed

Gingery, Anne; Bradley, Elizabeth; Shaw, Aubie; Oursler, Merry Jo

2003-05-01

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival. Copyright 2003 Wiley-Liss, Inc.

Response of Vitis vinifera cell cultures to Eutypa lata and Trichoderma atroviride culture filtrates: expression of defence-related genes and phenotypes.

PubMed

Mutawila, C; Stander, C; Halleen, F; Vivier, M A; Mostert, L

2017-03-01

Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

Paraquat initially damages cochlear support cells leading to anoikis-like hair cell death.

PubMed

Zhang, Jianhui; Sun, Hong; Salvi, Richard; Ding, Dalian

2018-07-01

Paraquat (PQ), one of the most widely used herbicides, is extremely dangerous because it generates the highly toxic superoxide radical. When paraquat was applied to cochlear organotypic cultures, it not only damaged the outer hair cells (OHCs) and inner hair cells (IHCs), but also caused dislocation of the hair cell rows. We hypothesized that the dislocation arose from damage to the support cells (SCs) that anchors hair cells within the epithelium. To test this hypothesis, rat postnatal cochlear cultures were treated with PQ. Shortly after PQ treatment, the rows of OHCs separated from one another and migrated radially away from IHCs suggesting loss of cell-cell adhesion that hold the hair cells in proper alignment. Hair cells dislocation was associated with extensive loss of SCs in the organ of Corti, loss of tympanic border cells (TBCs) beneath the basilar membrane, the early appearance of superoxide staining and caspase-8 labeling in SCs below the OHCs and disintegration of E-cadherin and β-catenin in the organ of Corti. Damage to the TBCs and SCs occurred prior to loss of OHC or IHC loss suggesting a form of detachment-induced apoptosis referred to as anoikis. Copyright © 2018 Elsevier B.V. All rights reserved.

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota

PubMed Central

Mourad, Elhussein F; Sarhan, Mohamed S; Daanaa, Hassan-Sibroe A; Abdou, Mennatullah; Morsi, Ahmed T; Abdelfadeel, Mohamed R; Elsawey, Hend; Nemr, Rahma; El-Tahan, Mahmoud; Hamza, Mervat A; Abbas, Mohamed; Youssef, Hanan H; Abdelhadi, Abdelhadi A; Amer, Wafaa M; Fayez, Mohamed; Ruppel, Silke; Hegazi, Nabil A

2018-01-01

In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S′, H′, and D′) based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies. PMID:29479006

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota.

PubMed

Mourad, Elhussein F; Sarhan, Mohamed S; Daanaa, Hassan-Sibroe A; Abdou, Mennatullah; Morsi, Ahmed T; Abdelfadeel, Mohamed R; Elsawey, Hend; Nemr, Rahma; El-Tahan, Mahmoud; Hamza, Mervat A; Abbas, Mohamed; Youssef, Hanan H; Abdelhadi, Abdelhadi A; Amer, Wafaa M; Fayez, Mohamed; Ruppel, Silke; Hegazi, Nabil A

2018-03-29

In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S', H', and D') based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors.

PubMed

Kropp, Christina; Kempf, Henning; Halloin, Caroline; Robles-Diaz, Diana; Franke, Annika; Scheper, Thomas; Kinast, Katharina; Knorpp, Thomas; Joos, Thomas O; Haverich, Axel; Martin, Ulrich; Zweigerdt, Robert; Olmer, Ruth

2016-10-01

: The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 10 6 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine. ©AlphaMed Press.

Lidocaine/monoethylglycinexylidide test, galactose elimination test, and sorbitol elimination test for metabolic assessment of liver cell bioreactors.

PubMed

Gerlach, Jörg C; Brayfield, Candace; Puhl, Gero; Borneman, Reiner; Müller, Christian; Schmelzer, Eva; Zeilinger, Katrin

2010-06-01

Various metabolic tests were compared for the performance characterization of a liver cell bioreactor as a routine function assessment of cultures in a standby for patient application in clinical studies. Everyday quality assessment (QA) is essential to ensure a continuous level of cellular functional capacity in the development of hepatic progenitor cell expansion systems providing cells for regenerative medicine research; it is also of interest to meet safety requirements in bioartificial extracorporeal liver support systems under clinical evaluation. Quality criteria for the description of bioreactor cultures were developed using primary porcine liver cells as a model. Porcine liver cells isolated by collagenase perfusion with an average of 3 x 10(9) primary cells were used in 39 bioreactors for culture periods up to 33 days. Measurements of monoethylglycinexylidide synthesis and elimination of lidocaine, galactose elimination, and sorbitol elimination proved to be useful for routine QA of primary liver cell cultures. We demonstrate two methods for dispensing test substances, bolus administration and continuous, steady-state administration. Bolus test data were grouped in Standard, Therapy, Infection/Contamination, and Cell-free control groups. Statistical analyses show significant differences among all groups for every test substance. Post hoc comparisons indicated significant differences between Standard and Cell-free groups for all elimination parameters. For continuous tests, results were categorized according to number of culture days and time-dependent changes were analyzed. Continuous administration enables a better view of culture health and the time dependency of cellular function, whereas bolus administration is more flexible. Both procedures can be used to define cell function. Assessment of cellular function and bioreactor quality can contribute significantly to the quality of experimental or clinical studies in the field of hepatic bioreactor development.

Effect of culture residence time on substrate uptake and storage by a pure culture of Thiothrix (CT3 strain) under continuous or batch feeding.

PubMed

Valentino, Francesco; Beccari, Mario; Villano, Marianna; Tandoi, Valter; Majone, Mauro

2017-05-25

A pure culture of the filamentous bacterium Thiothrix, strain CT3, was aerobically cultured in a chemostat under continuous acetate feeding at three different culture residence times (RT 6, 12 or 22 d) and the same volumetric organic load rate (OLR 0.12gCOD/L/d). Cells cultured at decreasing RT in the chemostat had an increasing transient response to acetate spikes in batch tests. The maximum specific acetate removal rate increased from 25 to 185mgCOD/gCOD/h, corresponding to a 1.8 to 8.1 fold higher respective steady-state rate in the chemostat. The transient response was mainly due to acetate storage in the form of poly(3-hydroxybutyrate) (PHB), whereas no growth response was observed at any RT. Interestingly, even though the storage rate also decreased as the RT increased, the storage yield increased from 0.41 to 0.50 COD/COD. This finding does not support the traditional view that storage plays a more important role as the transient response increases. The transient response of the steady-state cells was much lower than in cells cultured under periodic feeding (at 6 d RT, from 82 to 247mgCOD/gCOD/h), with the latter cells showing both storage and growth responses. On the other hand, even though steady-state cells had no growth response and their storage rate was also less, steady-state cells showed a higher storage yield than cells cultured under dynamic feeding. This suggests that in Thiothrix strain CT3, the growth response is triggered by periodic feeding, whereas the storage response is a constitutive mechanism, independent from previous acclimation to transient conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

PubMed Central

Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

PubMed Central

WATANABE, YUSAKU; YOSHIMURA, KIYOSHI; YOSHIKAWA, KOICHI; TSUNEDOMI, RYOICHI; SHINDO, YOSHITARO; MATSUKUMA, SOU; MAEDA, NORIKO; KANEKIYO, SHINSUKE; SUZUKI, NOBUAKI; KURAMASU, ATSUO; SONODA, KOUHEI; TAMADA, KOJI; KOBAYASHI, SEI; SAYA, HIDEYUKI; HAZAMA, SHOICHI; OKA, MASAAKI

2014-01-01

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy. PMID:25118635

Rat astrocytes are more supportive for mouse OPC self-renewal than mouse astrocytes in culture.

PubMed

Cheng, Xuejun; Xie, Binghua; Qi, Jiajun; Zhao, Xiaofeng; Zhang, Zunyi; Qiu, Mengsheng; Yang, Junlin

2017-09-01

Mouse primary oligodendrocyte precursor cells (OPCs) are increasingly used to study the molecular mechanisms underlying the phenotype changes in oligodendrocyte differentiation and axonal myelination observed in transgenic or mutant mouse models. However, mouse OPCs are much more difficult to be isolated by the simple dissociation culture of brain tissues than their rat counterparts. To date, the mechanisms underlying the species difference in OPC preparation remain obscure. In this study, we showed that astrocytes from rats have a stronger effect than those from mouse in promoting OPC proliferation and survival in vitro. Mouse astrocytes displayed significantly weaker viability in culture and reduced potential in maintaining OPC self-renewal, as confirmed by culturing OPCs with conditioned media from rat or mouse astrocytes. These results explained the reason for why stratified cultures of OPCs and astrocytes are difficult to be achieved in mouse CNS tissues. Based on these findings, we adopted inactivated rat astrocytes as feeder cells to support the self-renewal of mouse cortical OPCs and preparation of high-purity mouse OPCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 907-916, 2017. © 2016 Wiley Periodicals, Inc.

[Stem cell research and science and technology policy in Japan].

PubMed

Yashiro, Yoshimi

2011-12-01

In this paper I review the present condition of the regeneration medicine research using pluripotency and a somatic stem cell, and I describe the subject of the science and technology policy in Japan towards realization of regeneration medicine. The Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) supported research promotion by the prompt action in 2007 when establishment of the iPS cell was reported by Shinya Yamanaka. Although the hospitable support of the Japanese government to an iPS cell is continued still now, there are some problems in respect of the support to other stem cell researches, and industrialization of regeneration medicine. In order to win a place in highly competitive area of investigation, MEXT needs to change policy so that funds may be widely supplied also to stem cell researches other than iPS cell research.

Interplay of differential cell mechanical properties, motility, and proliferation in emergent collective behavior of cell co-cultures

NASA Astrophysics Data System (ADS)

Sutter, Leo; Kolbman, Dan; Wu, Mingming; Ma, Minglin; Das, Moumita

The biophysics of cell co-cultures, i.e. binary systems of cell populations, is of great interest in many biological processes including formation of embryos, and tumor progression. During these processes, different types of cells with different physical properties are mixed with each other, with important consequences for cell-cell interaction, aggregation, and migration. The role of the differences in their physical properties in their collective behavior remains poorly understood. Furthermore, until recently most theoretical studies of collective cell migration have focused on two dimensional systems. Under physiological conditions, however, cells often have to navigate three dimensional and confined micro-environments. We study a confined, three-dimensional binary system of interacting, active, and deformable particles with different physical properties such as deformability, motility, adhesion, and division rates using Langevin Dynamics simulations. Our findings may provide insights into how the differences in and interplay between cell mechanical properties, division, and motility influence emergent collective behavior such as cell aggregation and segregation experimentally observed in co-cultures of breast cancer cells and healthy breast epithelial cells. This work was partially supported by a Cottrell College Science Award.

A low-cost microwell device for high-resolution imaging of neurite outgrowth in 3D

NASA Astrophysics Data System (ADS)

Ren, Yuan; Mlodzianoski, Michael J.; Cheun Lee, Aih; Huang, Fang; Suter, Daniel M.

2018-06-01

Objective. Current neuronal cell culture is mostly performed on two-dimensional (2D) surfaces, which lack many of the important features of the native environment of neurons, including topographical cues, deformable extracellular matrix, and spatial isotropy or anisotropy in three dimensions. Although three-dimensional (3D) cell culture systems provide a more physiologically relevant environment than 2D systems, their popularity is greatly hampered by the lack of easy-to-make-and-use devices. We aim to develop a widely applicable 3D culture procedure to facilitate the transition of neuronal cultures from 2D to 3D. Approach. We made a simple microwell device for 3D neuronal cell culture that is inexpensive, easy to assemble, and fully compatible with commonly used imaging techniques, including super-resolution microscopy. Main results. We developed a novel gel mixture to support 3D neurite regeneration of Aplysia bag cell neurons, a system that has been extensively used for quantitative analysis of growth cone dynamics in 2D. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo. We demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. Significance. Neuronal cell culture has been a valuable tool for neuroscientists to study the behavior of neurons in a controlled environment. Compared to 2D, neurons cultured in 3D retain the majority of their native characteristics, while offering higher accessibility, control, and repeatability. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration.

Fabrication of micropatterned hydrogels for neural culture systems using dynamic mask projection photolithography.

PubMed

Curley, J Lowry; Jennings, Scott R; Moore, Michael J

2011-02-11

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities. Studies in fields such as cancer research, neural engineering, cardiac physiology, and cell-matrix interaction have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs. Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning, elastomer stamps, inkjet printing, additive photopatterning, static photomask projection-lithography, and dynamic mask microstereolithography. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates. Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (<200 μm) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization. In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Long Term Ex Vivo Culture and Live Imaging of Drosophila Larval Imaginal Discs.

PubMed

Tsao, Chia-Kang; Ku, Hui-Yu; Lee, Yuan-Ming; Huang, Yu-Fen; Sun, Yi Henry

Continuous imaging of live tissues provides clear temporal sequence of biological events. The Drosophila imaginal discs have been popular experimental subjects for the study of a wide variety of biological phenomena, but long term culture that allows normal development has not been satisfactory. Here we report a culture method that can sustain normal development for 18 hours and allows live imaging. The method is validated in multiple discs and for cell proliferation, differentiation and migration. However, it does not support disc growth and cannot support cell proliferation for more than 7 to 12 hr. We monitored the cellular behavior of retinal basal glia in the developing eye disc and found that distinct glia type has distinct properties of proliferation and migration. The live imaging provided direct proof that wrapping glia differentiated from existing glia after migrating to the anterior front, and unexpectedly found that they undergo endoreplication before wrapping axons, and their nuclei migrate up and down along the axons. UV-induced specific labeling of a single carpet glia also showed that the two carpet glia membrane do not overlap and suggests a tiling or repulsion mechanism between the two cells. These findings demonstrated the usefulness of an ex vivo culture method and live imaging.

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

PubMed

Omlor, G W; Nerlich, A G; Tirlapur, U K; Urban, J P; Guehring, T

2014-12-01

Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. 3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

Surface-Acoustic-Wave (SAW)-Driven Device for Dynamic Cell Cultures.

PubMed

Greco, Gina; Agostini, Matteo; Tonazzini, Ilaria; Sallemi, Damiano; Barone, Stefano; Cecchini, Marco

2018-06-19

In the last few decades, new types of cell cultures have been introduced to provide better cell survival and development, with micro- and nanoenvironmental physicochemical conditions aimed at mimicking those present in vivo. However, despite the efforts made, the systems available to date are often difficult to replicate and use. Here, an easy-to-use surface-acoustic-wave (SAW)-based platform is presented for realizing dynamic cell cultures that is compatible with standard optical microscopes, incubators, and cell-culture dishes. The SAW chip is coupled to a standard Petri dish via a polydimethylsiloxane (PDMS) disc and consists of a lithium niobate (LN) substrate on which gold interdigital transducers (IDTs) are patterned to generate the SAWs and induce acoustic streaming in the dish. SAW excitation is verified and characterized by laser Doppler vibrometry, and the fluid dynamics is studied by microparticle image velocimetry (μPIV). Heating is measured by an infrared (IR) thermal camera. We finally tested this device with the U-937 monocyte cell line for viability and proliferation and cell-morphological analysis. The data demonstrate that it is possible to induce significant fluid recirculation within the Petri dish while maintaining negligible heating. Remarkably, cell proliferation in this condition was enhanced by 36 ± 12% with respect to those of standard static cultures. Finally, we show that cell death does not increase and that cell morphology is not altered in the presence of SAWs. This device is the first demonstration that SAW-induced streaming can mechanically improve cell proliferation and further supports the great versatility and biocompatibility of the SAW technology for cell manipulation.

Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions.

PubMed

Daly, Andrew C; Sathy, Binulal N; Kelly, Daniel J

2018-01-01

Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O 2 and 3% O 2 . At 20% O 2 , dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O 2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.

[Proliferation and osteogenic differentiation of mesenchymal stem cells in hydrogels of human blood plasma].

PubMed

Linero, Itali M; Doncel, Adriana; Chaparro, Orlando

2014-01-01

The use of mesenchymal stem cells in clinical practice has increased considerably in the last decade because they play a supporting role in the processes of tissue repair and regeneration, becoming the main tool of cell therapy for the treatment of diseases functionally affecting bone and cartilage tissue . To evaluate in vitro the proliferative and osteogenic differentiation ability of mesenchymal stem cells derived from human adipose tissue in a blood plasma hydrogel. Mesenchymal stem cells were obtained from human adipose tissue explants and characterized by flow cytometry. Their multipotentiality was demonstrated by their ability to differentiate to adipogenic and osteogenic lineages. Cell proliferation and osteogenic differentiation ability of the cells cultured in blood plasma hydrogels were also evaluated. Mesenchymal stem cells derived from human adipose tissue growing in human blood plasma hydrogels showed a pattern of proliferation similar to that of the cells cultured in monolayer and also maintained their ability to differentiate to osteogenic lineage. Human blood plasma hydrogels are a suitable support for proliferation and osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue and provides a substrate that is autologous, biocompatible, reabsorbable, easy to use, potentially injectable and economic, which could be used as a successful strategy for the management and clinical application of cell therapy in regenerative medicine.

Engineering cartilaginous grafts using chondrocyte-laden hydrogels supported by a superficial layer of stem cells.

PubMed

Mesallati, Tariq; Buckley, Conor T; Kelly, Daniel J

2017-05-01

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co-cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self-assembled on top of CC-laden agarose gels (structured co-culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co-culture). Porcine infrapatellar fat pad-derived stem cells (FPSCs) and bone marrow-derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co-culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co-culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co-culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co-culture for both MSC sources. In conclusion, assembling MSCs onto CC-laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

PubMed

Miki, Hideo; Takagi, Mutsumi

2015-08-01

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

Support for the initial attachment, growth and differentiation of MG-63 cells: a comparison between nano-size hydroxyapatite and micro-size hydroxyapatite in composites

PubMed Central

Filová, Elena; Suchý, Tomáš; Sucharda, Zbyněk; Šupová, Monika; Žaloudková, Margit; Balík, Karel; Lisá, Věra; Šlouf, Miroslav; Bačáková, Lucie

2014-01-01

Hydroxyapatite (HA) is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N) or micro-sized (M) HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v) (referred to as N0–N25 or M0–M25), and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%–15%), MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%–15% (v/v) nano-size or micro-size HA seems to be optimum. PMID:25125978

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

PubMed

Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

2015-03-02

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.

Low-level laser irradiation modifies the effect of hyperglycemia on adhesion molecule levels.

PubMed

Góralczyk, Krzysztof; Szymańska, Justyna; Gryko, Łukasz; Fisz, Jacek; Rość, Danuta

2018-05-03

Endothelium plays a key role in maintaining vascular homeostasis by secreting active factors involved in many biological processes such as hemostasis, angiogenesis, and inflammation. Hyperglycemia in diabetic patients causes dysfunction of endothelial cells. Soluble fractions of adhesion molecules like sE-selectin and vascular cell adhesion molecule (sVCAM) are considered as markers of endothelial damage. The low-level laser therapy (LLLT) effectively supports the conventional treatment of vascular complications in diabetes, for example hard-to-heal wounds in patients with diabetic foot syndrome. The aim of our study was to evaluate the effect of low-energy laser at the wavelength of 635 nm (visible light) and 830 nm (infrared) on the concentration of adhesion molecules: sE-selectin and sVCAM in the supernatant of endothelial cell culture of HUVEC line. Cells were cultured under high-glucose conditions of 30 mM/L. We have found an increase in sE-selectin and sVCAM levels in the supernatant of cells cultured under hyperglycemic conditions. This fact confirms detrimental influence of hyperglycemia on vascular endothelial cell cultures. LLLT can modulate the inflammation process. It leads to a decrease in sE-selectin and sVCAM concentration in the supernatant and an increase in the number of endothelial cells cultured under hyperglycemic conditions. The influence of LLLT is greater at the wavelength of 830 nm.

Cell studies of hybridized carbon nanofibers containing bioactive glass nanoparticles using bone mesenchymal stromal cells

NASA Astrophysics Data System (ADS)

Zhang, Xiu-Rui; Hu, Xiao-Qing; Jia, Xiao-Long; Yang, Li-Ka; Meng, Qing-Yang; Shi, Yuan-Yuan; Zhang, Zheng-Zheng; Cai, Qing; Ao, Yin-Fang; Yang, Xiao-Ping

2016-12-01

Bone regeneration required suitable scaffolding materials to support the proliferation and osteogenic differentiation of bone-related cells. In this study, a kind of hybridized nanofibrous scaffold material (CNF/BG) was prepared by incorporating bioactive glass (BG) nanoparticles into carbon nanofibers (CNF) via the combination of BG sol-gel and polyacrylonitrile (PAN) electrospinning, followed by carbonization. Three types (49 s, 68 s and 86 s) of BG nanoparticles were incorporated. To understand the mechanism of CNF/BG hybrids exerting osteogenic effects, bone marrow mesenchymal stromal cells (BMSCs) were cultured directly on these hybrids (contact culture) or cultured in transwell chambers in the presence of these materials (non-contact culture). The contributions of ion release and contact effect on cell proliferation and osteogenic differentiation were able to be correlated. It was found that the ionic dissolution products had limited effect on cell proliferation, while they were able to enhance osteogenic differentiation of BMSCs in comparison with pure CNF. Differently, the proliferation and osteogenic differentiation were both significantly promoted in the contact culture. In both cases, CNF/BG(68 s) showed the strongest ability in influencing cell behaviors due to its fastest release rate of soluble silicium-relating ions. The synergistic effect of CNF and BG would make CNF/BG hybrids promising substrates for bone repairing.

Senescence-associated ultrastructural features of long-term cultures of induced pluripotent stem cells (iPSCs)

PubMed Central

Colasuonno, Fiorella; Borghi, Rossella; Niceforo, Alessia; Muzzi, Maurizio; Bertini, Enrico; Di Giulio, Andrea

2017-01-01

Induced pluripotent stem cells (iPSCs) hold great promise for developing personalized regenerative medicine, however characterization of their biological features is still incomplete. Moreover, changes occurring in long-term cultured iPSCs have been reported, suggesting these as a model of cellular aging. For this reason, we addressed the ultrastructural characterization of iPSCs, with a focus on possible time-dependent changes, involving specific cell compartments. To this aim, we comparatively analysed cultures at different timepoints, by an innovative electron microscopic technology (FIB/SEM). We observed progressive loss of cell-to-cell contacts, associated with increased occurrence of exosomes. Mitochondria gradually increased, while acquiring an elongated shape, with well-developed cristae. Such mitochondrial maturation was accompanied by their turnover, as assessed by the presence of autophagomes (undetectable in young iPSCs), some containing recognizable mitochondria. This finding was especially frequent in middle-aged iPSCs, while being occasional in aged cells, suggesting early autophagic activation followed by a decreased efficiency of the process with culturing time. Accordingly, confocal microscopy showed age-dependent alterations to the expression and distribution of autophagic markers. Interestingly, responsivity to rapamycin, highest in young iPSCs, was almost lost in aged cells. Overall, our results strongly support long-term cultured iPSCs as a model for studying relevant aspects of cellular senescence, involving intercellular communication, energy metabolism, and autophagy. PMID:29064821

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells.

PubMed

Konagaya, Shuhei; Iwata, Hiroo

2015-01-01

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress. hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons. Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved. hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads. Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons. Copyright © 2014 Elsevier B.V. All rights reserved.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

PubMed Central

Kropp, Christina; Kempf, Henning; Halloin, Caroline; Robles-Diaz, Diana; Franke, Annika; Scheper, Thomas; Kinast, Katharina; Knorpp, Thomas; Joos, Thomas O.; Haverich, Axel; Martin, Ulrich; Olmer, Ruth

2016-01-01

The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells’ energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs’ energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. Significance Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine. PMID:27369897

Development of scaffold architectures and heterotypic cell systems for hepatocyte transplantation

NASA Astrophysics Data System (ADS)

Alzebdeh, Dalia Abdelrahim

In vitro assembly of functional liver tissue is needed to enable the transplantation of tissue-engineered livers. In addition, there is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of sizable implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability, an issue which is closely related to the high metabolic rate of hepatocytes. In this study, we first compared two designs of highly porous chitosan-heparin scaffolds seeded with hepatocytes in dynamic perfusion bioreactor systems. The aim was to promote cell seeding efficiency by effectively entrapping 100 million hepatocytes at high density. We found that scaffolds with radially tapering pore architecture had highly efficient cell entrapment that maximized donor hepatocyte utilization, compared to alternate pore structures. Hepatocytes showed higher seeding efficiency and metabolic function when seeded as single cell suspensions as opposed to pre-formed, 100microm aggregates. Seeding efficiency was found to increase with flow rate, with single cell and aggregate suspension exhibiting different optimal flow rates. However, metabolic performance results indicated significant shear damage to cells at high efficiency flow rates. To better maintain hepatocyte basement membrane and cell polarity, spheroid co-cultures with mesenchymal stem cells (MSC) were investigated. Hepatocytes and MSCs were seeded in three different architectures in an effort to optimize the spatial arrangement of the two cell types. MSC co-culture greatly enhanced hepatocyte metabolic function in agitated cultures. Interestingly, the effects of diffusion limitations in spheroid culture, coupled with shear damage and subsequent removal of outer hepatocyte layers produced a defined oscillation of urea production rates in certain co-culture arrangements. A mathematical model of urea synthesis in shear-exposed, co-culture spheroids reproduced the metabolic oscillations observed. This result together with culture observations suggests that MSCs can provide both physiological support and some direct shear protection to hepatocytes in perfused or shear-exposed culture environments. Finally, in order to reduce hepatocyte exposure to excessive shear forces in perfused scaffolds, a modular scaffold design based on polyelectrolyte fiber encapsulation was explored. Scaffolds with uniformly distributed, shear protected cells were achieved.

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

PubMed Central

Braslavsky, Ido

2018-01-01

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices. PMID:29447224

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate.

PubMed

Bahari, Liat; Bein, Amir; Yashunsky, Victor; Braslavsky, Ido

2018-01-01

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.

Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI)

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. In this picture, the beads are trapped in the injection port shortly after injection. Swirls of beads indicate, event to the naked eye, the contents of the TCM are not fully mixed. The beads are similar in size and density to human lymphoid cells. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light

The biologic properties of recombinant human thrombopoietin in the proliferation and megakaryocytic differentiation of acute myeloblastic leukemia cells.

PubMed

Matsumura, I; Kanakura, Y; Kato, T; Ikeda, H; Horikawa, Y; Ishikawa, J; Kitayama, H; Nishiura, T; Tomiyama, Y; Miyazaki, H; Matsuzawa, Y

1996-10-15

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic level in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.

Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

PubMed Central

Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

2012-01-01

Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

Mechanisms of asbestos-induced squamous metaplasia in tracheobronchial epithelial cells.

PubMed Central

Cameron, G; Woodworth, C D; Edmondson, S; Mossman, B T

1989-01-01

Within 1 to 4 weeks after exposure to asbestos, differentiated rodent and human tracheobronchial epithelial cells in organ culture undergo squamous metaplasia, a putative preneoplastic lesion characterized by conversion of mucociliary cell types to keratinizing cells. The exogenous addition of retinal acetate (RA) to culture medium of hamster tracheal organ cultures reverses preestablished, asbestos-induced squamous metaplasia, although data suggest that the effectiveness of RA decreases as the length of time between exposure to asbestos and initial application of RA increases. alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), inhibits squamous metaplasia caused by asbestos or vitamin A deficiency, whereas addition of methylglyoxal bis(guanylhydrazone) (MGBG), a structural analog of spermidine and inhibitor of S-adenosylmethionine decarboxylase, causes an enhancement of metaplasia under both circumstances. Basal cell hyperplasia and increased incorporation of 3H-thymidine by tracheal epithelial cells also are seen after addition of the polyamines, putrescine or spermidine, to tracheal organ cultures, an observation supporting the importance of polyamines in the development of this lesion. The use of retinoids and inhibitors of ODC could be promising as preventive and/or therapeutic approaches for individuals at high risk for development of asbestos-associated diseases. PMID:2924752

Polyurethane acrylates as effective substrates for sustained in vitro culture of human myotubes.

PubMed

Andriani, Yosephine; Chua, Jason Min-Wen; Chua, Benjamin Yan-Jiang; Phang, In Yee; Shyh-Chang, Ng; Tan, Wui Siew

2017-07-15

Muscular disease has debilitating effects with severe damage leading to death. Our knowledge of muscle biology, disease and treatment is largely derived from non-human cell models, even though non-human cells are known to differ from human cells in their biochemical responses. Attempts to develop highly sought after in vitro human cell models have been plagued by early cell delamination and difficulties in achieving human myotube culture in vitro. In this work, we developed polyurethane acrylate (PUA) materials to support long-term in vitro culture of human skeletal muscle tissue. Using a constant base with modulated crosslink density we were able to vary the material modulus while keeping surface chemistry and roughness constant. While previous studies have focused on materials that mimic soft muscle tissue with stiffness ca. 12kPa, we investigated materials with tendon-like surface moduli in the higher 150MPa to 2.4GPa range, which has remained unexplored. We found that PUA of an optimal modulus within this range can support human myoblast proliferation, terminal differentiation and sustenance beyond 35days, without use of any extracellular protein coating. Results show that PUA materials can serve as effective substrates for successful development of human skeletal muscle cell models and are suitable for long-term in vitro studies. We developed polyurethane acrylates (PUA) to modulate the human skeletal muscle cell growth and maturation in vitro by controlling surface chemistry, morphology and tuning material's stiffness. PUA was able to maintain muscle cell viability for over a month without any detectable signs of material degradation. The best performing PUA prevented premature cell detachment from the substrate which often hampered long-term muscle cell studies. It also supported muscle cell maturation up to the late stages of differentiation. The significance of these findings lies in the possibility to advance studies on muscle cell biology, disease and therapy by using human muscle cells instead of relying on the widely used animal-based in vitro models. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells.

PubMed

Zhang, Wei; Hu, Jiabo; Ma, Quanhui; Hu, Sanqiang; Wang, Yanyan; Wen, Xiangmei; Ma, Yongbin; Xu, Hong; Qian, Hui; Xu, Wenrong

2014-09-01

An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at -70°C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 μg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 μg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at -70°C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird's nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at -70°C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with -70°C short-term or liquid nitrogen cryopreservation.

Megakaryocyte Polyploidization and Proplatelet Formation in Low-Attachment Conditions

PubMed Central

Schlinker, Alaina C.; Whitehead, David C.; Miller, William M.

2016-01-01

In vitro-derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture. PMID:27087780

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

PubMed

Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

2016-09-01

Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. None. This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

PubMed Central

Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.

2016-01-01

STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. PMID:27430551

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

PubMed

Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function: Implications for Off-The Shelf ES-MSC Therapies

PubMed Central

Li, Ou; Tormin, Ariane; Sundberg, Berit; Hyllner, Johan; Le Blanc, Katarina; Scheding, Stefan

2013-01-01

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function. PMID:23383153

Three-dimensional hydrogel cell culture systems for modeling neural tissue

NASA Astrophysics Data System (ADS)

Frampton, John

Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was designed for use as a tool to predict the transport and processing that occurs prior to drug uptake in the central nervous system (CNS), and to predict BBB permeability. Electrochemical techniques and immunohistochemistry were used to validate this model and provide detailed information about cellular organization and function. Electrochemical impedance spectroscopy (EIS) provided evidence that endothelial cells cultured in the presence of astrocytes formed tight junctions capable of occluding the flow of electrical current. In a second series of experiments, a microglia-astrocyte co-culture system was developed to assess the effects of glial cells on electrode impedance recorded from neural prosthetic devices in vitro. Impedance measurements were compared with confocal images to determine the effects of glial cell density and cell type on electrode performance. The results indicate that EIS data can be used to model components of the reactive cell responses in brain tissue, and that impedance measurements recorded in vitro can be compared to measurements recorded in vivo. Taken together, these results demonstrate that alginate hydrogels can be used for the creation of 3-D neural cell scaffolds, and that such cell scaffolds can be used to model a variety of three-dimensional neural tissues in vitro, that cannot be studied in 2-D cultures.

In vitro 3D regeneration-like growth of human patient brain tissue.

PubMed

Tang-Schomer, M D; Wu, W B; Kaplan, D L; Bookland, M J

2018-05-01

In vitro culture of primary neurons is widely adapted with embryonic but not mature brain tissue. Here, we extended a previously developed bioengineered three-dimensional (3D) embryonic brain tissue model to resected normal patient brain tissue in an attempt to regenerate human neurons in vitro. Single cells and small sized (diameter < 100 μm) spheroids from dissociated brain tissue were seeded into 3D silk fibroin-based scaffolds, with or without collagen or Matrigel, and compared with two-dimensional cultures and scaffold-free suspension cultures. Changes of cell phenotypes (neuronal, astroglial, neural progenitor, and neuroepithelial) were quantified with flow cytometry and analyzed with a new method of statistical analysis specifically designed for percentage comparison. Compared with a complete lack of viable cells in conventional neuronal cell culture condition, supplements of vascular endothelial growth factor-containing pro-endothelial cell condition led to regenerative growth of neurons and astroglial cells from "normal" human brain tissue of epilepsy surgical patients. This process involved delayed expansion of Nestin+ neural progenitor cells, emergence of TUJ1+ immature neurons, and Vimentin+ neuroepithelium-like cell sheet formation in prolonged cultures (14 weeks). Micro-tissue spheroids, but not single cells, supported the brain tissue growth, suggesting importance of preserving native cell-cell interactions. The presence of 3D scaffold, but not hydrogel, allowed for Vimentin+ cell expansion, indicating a different growth mechanism than pluripotent cell-based brain organoid formation. The slow and delayed process implied an origin of quiescent neural precursors in the neocortex tissue. Further optimization of the 3D tissue model with primary human brain cells could provide personalized brain disease models. Copyright © 2018 John Wiley & Sons, Ltd.

Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.

PubMed

Sharma, Ruchi; George, Aman; Kamble, Nitin Manchindra; Singh, Karn Pratap; Chauhan, Manmohan Singh; Singla, Suresh Kumar; Manik, Radhey Sham; Palta, Prabhat

2011-12-01

A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

Probiotic metabolites from Bacillus coagulans GanedenBC30TM support maturation of antigen-presenting cells in vitro

PubMed Central

Benson, Kathleen F; Redman, Kimberlee A; Carter, Steve G; Keller, David; Farmer, Sean; Endres, John R; Jensen, Gitte S

2012-01-01

AIM: To study the effects of probiotic metabolites on maturation stage of antigen-presenting immune cells. METHODS: Ganeden Bacillus coagulans 30 (GBC30) bacterial cultures in log phase were used to isolate the secreted metabolite (MET) fraction. A second fraction was made to generate a crude cell-wall-enriched fraction, by centrifugation and lysis, followed by washing. A preparation of MET was subjected to size exclusion centrifugation, generating three fractions: < 3 kDa, 3-30 kDa, and 30-200 kDa and activities were tested in comparison to crude MET and cell wall in primary cultures of human peripheral blood mononuclear cell (PBMC) as a source of antigen-presenting mononuclear phagocytes. The maturation status of mononuclear phagocytes was evaluated by staining with monoclonal antibodies towards CD14, CD16, CD80 and CD86 and analyzed by flow cytometry. RESULTS: Treatment of PBMC with MET supported maturation of mononuclear phagocytes toward both macrophage and dendritic cell phenotypes. The biological activity unique to the metabolites included a reduction of CD14+ CD16+ pro-inflammatory cells, and this property was associated with the high molecular weight metabolite fraction. Changes were also seen for the dendritic cell maturation markers CD80 and CD86. On CD14dim cells, an increase in both CD80 and CD86 expression was seen, in contrast to a selective increase in CD86 expression on CD14bright cells. The co-expression of CD80 and CD86 indicates effective antigen presentation to T cells and support of T helper cell differentiation. The selective expression of CD86 in the absence of CD80 points to a role in generating T regulatory cells. CONCLUSION: The data show that a primary mechanism of action of GBC30 metabolites involves support of more mature phenotypes of antigen-presenting cells, important for immunological decision-making. PMID:22563167

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

PubMed

Du, Xiaohui; Li, Wanming; Du, Guansheng; Cho, Hansang; Yu, Min; Fang, Qun; Lee, Luke P; Fang, Jin

2018-03-06

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

Growth Factors Polymerized Within Fibrin Hydrogel Promote Amylase Production in Parotid Cells

PubMed Central

McCall, Andrew D.; Nelson, Joel W.; Leigh, Noel J.; Duffey, Michael E.; Lei, Pedro; Andreadis, Stelios T.

2013-01-01

Salivary gland cell differentiation has been a recurring challenge for researchers as primary salivary cells show a loss of phenotype in culture. Particularly, parotid cells show a marked decrease in amylase expression, the loss of tight junction organization and proper cell function. Previously, Matrigel has been used successfully as an extracellular matrix; however, it is not practical for in vivo applications as it is tumorigenic. An alternative method could rely on the use of fibrin hydrogel (FH), which has been used extensively in biomedical engineering applications ranging from cardiovascular tissue engineering to wound-healing experiments. Although several groups have examined the effects of a three-dimensional (3D) environment on salivary cell cultures, little is known about the effects of FH on salivary cell cultures. The current study developed a 3D cell culture model to support parotid gland cell differentiation using a combination of FH and growth factor-reduced Matrigel (GFR-MG). Furthermore, FH polymerized with a combination of EGF and IGF-1 induced formation of 3D spheroids capable of amylase expression and an agonist-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) in salivary cells. These studies represent an initial step toward the construction of an artificial salivary gland to restore salivary gland dysfunction. This is necessary to reduce xerostomia in patients with compromised salivary function. PMID:23594102

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

PubMed Central

Creek, Darren J.; Nijagal, Brunda; Kim, Dong-Hyun; Rojas, Federico; Matthews, Keith R.

2013-01-01

In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening. PMID:23571546

Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation.

PubMed

Chastain, Sara R; Kundu, Anup K; Dhar, Sanjay; Calvert, Jay W; Putnam, Andrew J

2006-07-01

The osteogenic potential of mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA) or poly(caprolactone) (PCL), two widely used polymeric biomaterials that have been reported to differentially support osteogenic differentiation, was compared in these studies. Here we report that MSCs cultured in 3-D PLGA scaffolds for up to 5 weeks significantly upregulate osteocalcin gene expression levels. By contrast, osteocalcin expression was markedly downregulated in 3-D PCL-based constructs over the same time course. We hypothesized that differential adsorption of extracellular matrix (ECM) proteins present in serum-containing culture medium and subsequent differences in integrin-mediated adhesion are responsible for these differences, and tested this hypothesis using thin (2-D) polymeric films. Supporting this hypothesis, significant amounts of fibronectin and vitronectin deposited onto both materials in serum-containing osteogenic media, with type-I collagen present in lower amounts. Adhesion-blocking studies revealed that MSCs adhere to PCL primarily via vitronectin, while type-I collagen mediates their attachment to PLGA. These adhesive mechanisms correlated with higher levels of alkaline phosphatase (ALP) activity after 2 weeks of monolayer culture on PLGA versus PCL. These data suggest that the initial adhesion of MSCs to PLGA via type-I collagen fosters osteogenesis while adhesion to PCL via vitronectin does not, and stress the need for an improved molecular understanding of cell-ECM interactions in stem cell-based therapies. Copyright (c) 2006 Wiley Periodicals, Inc.

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

PubMed

Linning, Katrina D; Tai, Mei-Hui; Madhukar, Burra V; Chang, C C; Reed, Donald N; Ferber, Sarah; Trosko, James E; Olson, L Karl

2004-10-01

The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

Media composition: salts and osmolality.

PubMed

Baltz, Jay M

2012-01-01

The main components of embryo culture media are salts, which dissociate into their component inorganic ions in aqueous solution. All embryo culture media contain the same six inorganic ions: Na(+), K(+), Cl(-), Ca(2+), Mg(2+), and SO(4)(2-), while most also contain PO(4)(2-). The salts that are used to formulate embryo culture media can be traced back to classic saline solutions, particularly Krebs-Ringer Bicarbonate (KRB), that were developed for somatic cells in the first half of the twentieth century. The salt and inorganic ion concentrations in the first successful defined mouse embryo culture medium, Whittens medium, were identical to those in KRB. These remained largely unchanged in embryo culture media for decades, with similar levels found in the standard mouse embryo culture medium, M16, formulated in the 1970s. Human embryos were initially cultured in undefined somatic cell media such as Earles and Hams F-10 with serum added. This changed in the mid-1980s, however, with the development of Quinns HTF, a defined medium specifically formulated for human embryo culture, in which the inorganic ion concentrations are similar to those in M16 and Whittens. While these media were useful both for experimental work and clinically, embryos suffered developmental blocks in all of them, with mouse embryos blocking at the 2-cell stage and human embryos at the 4- to 8-cell stage. Starting in the late 1980s, however, mouse embryo culture media were first developed that alleviated these developmental blocks. These media, CZB and KSOM, had much lower osmolalities than previous media, mainly due to lower inorganic ion concentrations. Indeed, lowering total inorganic ion concentration and osmolality proved key to understanding how media that supported complete preimplantation development in vitro can be formulated. A subsequent improvement was the addition of amino acids to culture media for both mouse and human embryos. At least in part, their beneficial effect during the cleavage stages of development is due to the presence in early preimplantation embryos of mechanisms for cell volume regulation that depend on the accumulation of amino acids as organic osmolytes to provide intracellular osmotic support. These amino acids, principally glycine, replace a portion of the intracellular inorganic ions that would otherwise be needed to maintain cell size, preventing the intracellular ionic strength from rising to deleterious levels and blocking development. Thus, the optimum salts levels, osmolality, and amino acid contents of culture media are not independent, but interact strongly because of their roles in cell volume regulation. In the absence of compounds that preimplantation embryos can use as organic osmolytes, embryos will develop only at lower osmolalities and salt concentrations in the medium. However, when organic osmolytes such as some amino acids are present, embryos will develop in culture at higher osmolarities that are similar to those they experience in tubal fluid in vivo.

Engineered three-dimensional multicellular culture model to ...

EPA Pesticide Factsheets

Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in developmental processes including heart development, neural tube closure, and palatal fusion are dependent on epithelial-mesenchymal interactions (EMIs) and specific signaling pathways that have been elucidated largely using gene knockout mouse models. A broad analysis of literature using ToxRefDB identified 63 ToxCast chemicals associated with cleft palate in animal models. However, the influence of these and other putative teratogens on human palatal fusion has not been examined in depth due to the lack of in vitro models incorporating EMIs between human cell types. We sought to engineer the stratified mesenchymal and epithelial structure of the developing palate in vitro using spheroid culture of human Wharton’s Jelly mesenchymal stem cells (hMSC). hMSC spheroids exhibited uniform size over time (175 ± 21 µm mean diameter) that was proportional to starting cell density. Further, hMSCs in spheroid culture exhibited increased alkaline phosphatase activity and increased expression of bglap and runx2 after 7 days of culture in osteo-induction medium, which suggests that spheroid culture together with osteo-induction medium supports osteogenic differentiation. We developed a novel pro

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

PubMed

Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

2017-03-01

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

A 3D analysis of oxygen transfer in a low-cost micro-bioreactor for animal cell suspension culture.

PubMed

Yu, P; Lee, T S; Zeng, Y; Low, H T

2007-01-01

A 3D numerical model was developed to study the flow field and oxygen transport in a micro-bioreactor with a rotating magnetic bar on the bottom to mix the culture medium. The Reynolds number (Re) was kept in the range of 100-716 to ensure laminar environment for animal cell culture. The volumetric oxygen transfer coefficient (k(L)a) was determined from the oxygen concentration distribution. It was found that the effect of the cell consumption on k(L)a could be negligible. A correlation was proposed to predict the liquid-phase oxygen transfer coefficient (k(Lm)) as a function of Re. The overall oxygen transfer coefficient (k(L)) was obtained by the two-resistance model. Another correlation, within an error of 15%, was proposed to estimate the minimum oxygen concentration to avoid cell hypoxia. By combination of the correlations, the maximum cell density, which the present micro-bioreactor could support, was predicted to be in the order of 10(12) cells m(-3). The results are comparable with typical values reported for animal cell growth in mechanically stirred bioreactors.

Heparin affinity purification of extracellular vesicles

PubMed Central

Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

2015-01-01

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

PubMed

Praveen, Wulligundam; Madathil, Bernadette K; Sajin Raj, R S; Kumary, T V; Anil Kumar, P R

2017-10-25

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates

PubMed Central

Sip, Christopher G.; Bhattacharjee, Nirveek; Folch, Albert

2015-01-01

Gradients of biochemical molecules play a key role in many physiological processes such as axon growth, tissue morphogenesis, and trans-epithelium nutrient transport, as well as in pathophysiological phenomena such as wound healing, immune response, bacterial invasion, and cancer metastasis. In this paper, we report a microfluidic transwell insert for generating quantifiable concentration gradients in a user-friendly and modular format that is compatible with conventional cell cultures and with tissue explant cultures. The device is simply inserted into a standard 6-well plate, where it hangs self-supported at a distance of ~250 μm above the cell culture surface. The gradient is created by small microflows from the device, through an integrated track-etched porous membrane, into the cell culture well. The microfluidic transwell can deliver stable, quantifiable gradients over a large area with extremely low fluid shear stress to dissociated cells or tissue explants cultured independently on the surface of a 6-well plate. We used finite-element modeling to describe the porous membrane flow and molecular transport and to predict gradients generated by the device. Using the device, we applied a gradient of the chemotactic peptide N-Formyl-Met-Leu-Phe (fMLP) to a large population of HL-60 cells (a neutrophil cell line) and directly observed the migration with time-lapse microscopy. On quantification of the chemotactic response with an automated tracking algorithm, we found 74% of the cells moving towards the gradient. Additionally, the modular design and low fluid shear stress made it possible to apply gradients of growth factors and second messengers to mouse retinal explant cultures. With a simplified interface and well-defined gradients, the microfluidic transwell device has potential for broad applications to gradient-sensing biology. PMID:24225908

Hemoglobin Regulates the Metabolic, Synthetic, Detoxification, and Biotransformation Functions of Hepatoma Cells Cultured in a Hollow Fiber Bioreactor

PubMed Central

Chen, Guo

2010-01-01

Hepatic hollow fiber (HF) bioreactors constitute one type of extracorporeal bioartificial liver assist device (BLAD). Ideally, cultured hepatocytes in a BLAD should closely mimic the in vivo oxygenation environment of the liver sinusoid to yield a device with optimal performance. However, most BLADs, including hepatic HF bioreactors, suffer from O2 limited transport toward cultured hepatocytes, which reduces their performance. We hypothesize that supplementation of hemoglobin-based O2 carriers into the circulating cell culture medium of hepatic HF bioreactors is a feasible and effective strategy to improve bioreactor oxygenation and performance. We examined the effect of bovine hemoglobin (BvHb) supplementation (15 g/L) in the circulating cell culture medium of hepatic HF bioreactors on hepatocyte proliferation, metabolism, and varied liver functions, including biosynthesis, detoxification, and biotransformation. It was observed that BvHb supplementation supported the maintenance of a higher cell mass in the extracapillary space, improved hepatocyte metabolic efficiency (i.e., hepatocytes consumed much less glucose), improved hepatocyte capacity for drug metabolism, and conserved both albumin synthesis and ammonia detoxification functions compared to controls (no BvHb supplementation) under the same experimental conditions. PMID:20528678

Ectodermal fragments from normal frog gastrulae condition substrata to support normal and hybrid mesodermal cell migration in vitro.

PubMed

Nakatsuji, N; Johnson, K E

1984-06-01

Using time-lapse cinemicrography and scanning electron microscopy, we have shown that normal Rana embryos and gastrulating hybrid embryos have extracellular fibrils on the inner surface of the ectodermal layer. These fibrils are absent prior to gastrulation and appear in increasing numbers during gastrulation. They can also be deposited in vitro where they condition substrata in such a way that normal presumptive mesodermal cells placed on them show extensive attachment and unoriented cell movement. These fibrils are also present in some arrested hybrid embryos, but in reduced numbers, or are lacking in other arrested hybrid embryos. Explanted ectodermal fragments from arrested hybrid embryos fail both to condition culture substrata by the deposition of fibrils and to promote cell attachment and translocation. In contrast, ectodermal fragments from normal embryos can condition culture substrata so as to promote moderate cell attachment and, for one particular gamete combination, even cell translocation of presumptive mesodermal cells taken from arrested hybrid embryos. These results provide new evidence to support the hypothesis that extracellular fibrils represent a system that promotes mesodermal cell migration in amphibian embryos. Differences in the fibrillar system in urodele and anuran embryos are discussed in relation to fundamental differences in the mode of mesodermal cell migration in these two classes of Amphibia.

Identification of various cell culture models for the study of Zika virus

PubMed Central

Himmelsbach, Kiyoshi; Hildt, Eberhard

2018-01-01

AIM To identify cell culture models supportive for Zika virus (ZIKV) replication. METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis. RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5Y released 100 times less infectious viral particles than Vero-, A549- or 293T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines. CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research. PMID:29468137

Bioavailability of iron from spinach using an in vitro/human Caco-2 cell bioassay model

NASA Technical Reports Server (NTRS)

Rutzke, Corinne J.; Glahn, Raymond P.; Rutzke, Michael A.; Welch, Ross M.; Langhans, Robert W.; Albright, Louis D.; Combs, Gerald F Jr; Wheeler, Raymond M.

2004-01-01

Spinach (Spinacia oleracea) cv Whitney was tested for iron bioavailabilty using an in vitro human intestinal cell culture ferritin bioassay technique previously developed. Spinach was cultured in a growth chamber for 33 days, harvested, and freeze-dried. Total iron in the samples was an average of 71 micrograms/g dry weight. Spinach was digested in vitro (pepsin and 0.1 M HCl followed by pancreatin and 0.1 M NaHCO3) with and without the addition of supplemental ascorbic acid. Caco-2 cell cultures were used to determine iron bioavailability from the spinach mixtures. Production of the iron-binding protein ferritin in the Caco-2 cells showed the supplemental ascorbic acid doubled bioavailability of iron from spinach. The data show fresh spinach is a poor source of iron, and emphasize the importance of evaluation of whole meals rather than single food items. The data support the usefulness of the in vitro/Caco-2 cell ferritin bioassay model for prescreening of space flight diets for bioavailable iron.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

PubMed

Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

2015-01-01

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells.

PubMed

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

2012-06-22

Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-β in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-α, G-CSF, and TGF-β. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function. Copyright © 2012 Elsevier Inc. All rights reserved.

Production of oncolytic adenovirus and human mesenchymal stem cells in a single-use, Vertical-Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier-based cell culture processes.

PubMed

Sousa, Marcos F Q; Silva, Marta M; Giroux, Daniel; Hashimura, Yas; Wesselschmidt, Robin; Lee, Brian; Roldão, António; Carrondo, Manuel J T; Alves, Paula M; Serra, Margarida

2015-01-01

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers.

Wnt Responsive Lgr5-Expressing Stem Cells Are Hair Cell Progenitors in the Cochlea

PubMed Central

Shi, Fuxin; Kempfle, Judith; Edge, Albert S. B.

2012-01-01

Auditory hair cells are surrounded on their basolateral aspects by supporting cells, and these two cell types together constitute the sensory epithelium of the organ of Corti, which is the hearing apparatus of the ear. We show here that Lgr5, a marker for adult stem cells, was expressed in a subset of supporting cells in the newborn and adult murine cochlea. Lgr5-expressing supporting cells, sorted by flow cytometry and cultured in a single cell suspension, as compared to unsorted cells, displayed an enhanced capacity for self-renewing neurosphere formation in response to Wnt and were converted to hair cells at a higher (>10-fold) rate. The greater differentiation of hair cell in the neurosphere assay showed that Lgr5-positive cells had the capacity to act as cochlear progenitor cells, and lineage tracing confirmed that Lgr5-expressing cells accounted for the cells that formed neurospheres and differentiated to hair cells. The responsiveness to Wnt of cells with a capacity for division and sensory cell formation suggests a potential route to new hair cell generation in the adult cochlea. PMID:22787049

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

PubMed

Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

2018-02-01

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

PubMed

Lamas, Nuno Jorge; Johnson-Kerner, Bethany; Roybon, Laurent; Kim, Yoon A; Garcia-Diaz, Alejandro; Wichterle, Hynek; Henderson, Christopher E

2014-01-01

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

PubMed Central

Lamas, Nuno Jorge; Johnson-Kerner, Bethany; Roybon, Laurent; Kim, Yoon A.; Garcia-Diaz, Alejandro; Wichterle, Hynek; Henderson, Christopher E.

2014-01-01

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC50 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening. PMID:25337699

Count of splenic stromal precursor cells in mice and expression of cytokine genes in these cells in primary cultures during different periods after immunization of animals with S. typhimurium antigens.

PubMed

Gorskaya, Yu F; Danilova, T A; Mezentseva, M V; Shapoval, I M; Narovlyanskii, A N; Nesterenko, V G

2011-06-01

Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1β (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.

A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening

PubMed Central

Rijal, Girdhari; Li, Weimin

2017-01-01

Most of the anticancer drug candidates entering preclinical trials fail to be approved for clinical applications. The following are among the main causes of these failures: studying molecular mechanisms of cancer development, identifying therapeutic targets, and testing drug candidates using inappropriate tissue culture models, which do not recapitulate the native microenvironment where the cancer cells originate. It has become clear that three-dimensional (3D) cell cultures are more biologically and clinically relevant than 2D models. The spatial and mechanical conditions of 3D cultures enable the cancer cells to display heterogeneous growth, assume diverse phenotypes, express distinct gene and protein products, and attain metastatic potential and resistance to drugs that are reminiscent of tumors in humans. However, the current 3D culture systems using synthetic polymers or selected components of the extracellular matrix (ECM) are defective (particularly the biophysical and biochemical properties of the native ECM) and remain distant to optimally support the signaling cue–oriented cell survival and growth. We introduce a reconstitutable tissue matrix scaffold (TMS) system fabricated using native tissue ECM, with tissue-like architecture and resilience. The structural and compositional properties of TMS favor robust cell survival, proliferation, migration, and invasion in culture and vascularized tumor formation in animals. The combination of porous and hydrogel TMS allows compartmental culture of cancerous and stromal cells, which are distinguishable by biomarkers. The response of the cancer cells grown on TMS to drugs well reflects animal and clinical observations. TMS enables more biologically relevant studies and is suitable for preclinical drug screening. PMID:28924608

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PubMed

Chen, D; Qu, Y; Hua, X; Zhang, L; Liu, Z; Pflugfelder, S C; Li, D-Q

2017-06-01

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

Zinc oxide nanostructures as low-cost templates for neuronal circuit

NASA Astrophysics Data System (ADS)

Kritharidou, A.; Georgoussi, Z.; Tsamis, C.; Makarona, E.

2013-05-01

ZnO nanostructures were explored as templates for the development of topography-mediated neuronal cultures. Nanostructures of varying features were produced on 4" Si substrates via a rapid, facile and low-cost technique that allows the systematic investigation of nanotopographically-mediated formation of neuronal cultures. The developed ZnO-nanowire based templates were seeded with Neuro-2A mouse neuroblastoma cells and their viability over the course of 1 to 4 days was assessed. Our studies demonstrate that the ZnO-templates can support neuronal cell growth and proliferation suggesting that ZnO substrate can be used for the development of neuronal cell-based platform technologies.

Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures

PubMed Central

Myers, Jeremy N.; Rekhadevi, Perumalla V.; Ramesh, Aramandla

2011-01-01

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies. PMID:21865728

Control of cell growth on 3D-printed cell culture platforms for tissue engineering.

PubMed

Tan, Zhikai; Liu, Tong; Zhong, Juchang; Yang, Yikun; Tan, Weihong

2017-12-01

Biocompatible tissue growth has excellent prospects for tissue engineering. These tissues are built over scaffolds, which can influence aspects such as cell adhesion, proliferation rate, morphology, and differentiation. However, the ideal 3D biological structure has not been developed yet. Here, we applied the electro-hydrodynamic jet (E-jet) 3D printing technology using poly-(lactic-co-glycolic acid, PLGA) solution to print varied culture platforms for engineered tissue structures. The effects of different parameters (electrical voltage, plotting speed, and needle sizes) on the outcome were investigated. We compared the biological compatibility of the 3D printed culture platforms with that of random fibers. Finally, we used the 3D-printed PLGA platforms to culture fibroblasts, the main cellular components of loose connective tissue. The results show that the E-jet printed platforms could guide and improve cell growth. These highly aligned fibers were able to support cellular alignment and proliferation. Cell angle was consistent with the direction of the fibers, and cells cultured on these fibers showed a much faster migration, potentially enhancing wound healing performance. Thus, the potential of this technology for 3D biological printing is large. This process can be used to grow biological scaffolds for the engineering of tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3281-3292, 2017. © 2017 Wiley Periodicals, Inc.

Protective Effects of Bacopa Monnieri on Hydrogen Peroxide and Staurosporine: Induced Damage of Human Neuroblastoma SH-SY5Y Cells.

PubMed

Łojewski, Maciej; Pomierny, Bartosz; Muszyńska, Bożena; Krzyżanowska, Weronika; Budziszewska, Bogusława; Szewczyk, Agnieszka

2016-02-01

Many herbs, and recently their biomass from in vitro cultures, are essential for the treatment of diseases. The aim of this study was to determine the optimal growth of Bacopa monnieri (water hyssop) in an in vitro culture and to examine if extracts of the B. monnieri biomass from the in vitro culture would affect hydrogen peroxide- and staurosporine-induced injury of the human neuroblastoma SH-SY5Y cell line. It has been found that B. monnieri at concentrations of 25, 50, and 100 µg/mL inhibited both hydrogen peroxide-induced efflux of lactate dehydrogenase from damaged cells to culture medium and increased cell viability determined by an MTT assay. Moreover, B. monnieri at concentrations of 10, 25, and 50 µg/mL decreased staurosporine-induced activity of an executive apoptotic enzyme-caspase-3 and protected mitochondrial membrane potential. The obtained data indicate that the biomass from the in vitro culture of B. monnieri prevented SH-SY5Y cell damage related to oxidative stress and had the ability to inhibit the apoptotic process. Thus, this study supports the traditional use of B. monnieri as a neuroprotective therapy, and further in vivo studies on the effects of this preparation on morphology and function of nerve cells could lead to its wider application. Georg Thieme Verlag KG Stuttgart · New York.

A Novel In Vitro Model for Studying Quiescence and Activation of Primary Isolated Human Myoblasts

PubMed Central

Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna; Schrøder, Henrik Daa

2013-01-01

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts. PMID:23717533

Kidney Cell Electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

PubMed Central

Wise, Joel K.; Alford, Andrea I.; Goldstein, Steven A.

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25×106 cells/mL, containing an estimated 5×104 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2×105 cells/mL) were added to a 65–35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering. PMID:23879621

Comparison of uncultured marrow mononuclear cells and culture-expanded mesenchymal stem cells in 3D collagen-chitosan microbeads for orthopedic tissue engineering.

PubMed

Wise, Joel K; Alford, Andrea I; Goldstein, Steven A; Stegemann, Jan P

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 × 10(6) cells/mL, containing an estimated 5 × 10(4) MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 × 10(5) cells/mL) were added to a 65-35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.

Method for culturing mammalian cells in a perfused bioreactor

NASA Technical Reports Server (NTRS)

Schwarz, Ray P. (Inventor); Wolf, David A. (Inventor)

1992-01-01

A bio-reactor system wherein a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop.

Cryptococcus laurentii diarrhea post hematopoietic stem cell transplant.

PubMed

Bhat, Vivek; Vira, Hemant; Khattry, Navin; Toshniwal, Manoj

2017-04-01

We report the recent isolation of Cryptococcus laurentii from the feces of a patient with Hodgkin's lymphoma who underwent autologous hematopoietic stem cell transplant (HSCT). The organism was identified using microscopic morphology, cultural characteristics, and biochemical tests including sugar assimilation. Minimum inhibitory concentration of various antifungals was determined by microbroth dilution method. The recovery of pure culture of C. laurentii from stool culture, and the patient's response to treatment with voriconazole support its potential etiological role. To the best of our knowledge, we report the first case of diarrhea caused by C. laurentii in an HSCT recipient. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rotating bio-reactor cell culture apparatus

NASA Technical Reports Server (NTRS)

Schwarz, Ray P. (Inventor); Wolf, David A. (Inventor)

1991-01-01

A bioreactor system is described in which a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop.

Microfluidic chemostat and turbidostat with flow rate, oxygen, and temperature control for dynamic continuous culture.

PubMed

Lee, Kevin S; Boccazzi, Paolo; Sinskey, Anthony J; Ram, Rajeev J

2011-05-21

This work reports on an instrument capable of supporting automated microscale continuous culture experiments. The instrument consists of a plastic-PDMS device capable of continuous flow without volume drift or evaporation. We apply direct computer controlled machining and chemical bonding fabrication for production of fluidic devices with a 1 mL working volume, high oxygen transfer rate (k(L)a≈0.025 s(-1)), fast mixing (2 s), accurate flow control (±18 nL), and closed loop control over temperature, cell density, dissolved oxygen, and pH. Integrated peristaltic pumps and valves provide control over input concentrations and allow the system to perform different types of cell culture on a single device, such as batch, chemostat, and turbidostat continuous cultures. Continuous cultures are demonstrated without contamination for 3 weeks in a single device and both steady state and dynamically controlled conditions are possible. © The Royal Society of Chemistry 2011

Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation.

PubMed

Shen, Francis H; Werner, Brian C; Liang, Haixiang; Shang, Hulan; Yang, Ning; Li, Xudong; Shimer, Adam L; Balian, Gary; Katz, Adam J

2013-01-01

Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning. Multicellular aggregate cells expressed increased differentiation potential and extracellular matrix production over the same human ADS cells cultured in monolayer. Furthermore, MAs reseeded onto monolayer retained their stem cell capabilities. When implanted in vivo, significantly greater bone volume and extracellular matrix were present in the implanted specimens of MAs confirmed on both microCT and histological sectioning. This is the first study to investigate the capability of human ADS cells in a 3D culture system to undergo osteogenic differentiation. The results confirm that MAs maintain their stem cell characteristics. Compared with analogous cells in monolayer culture, the human ADS cells as MAs exhibit elevated levels of osteogenic differentiation and increased matrix mineralization. Furthermore, the creation of uniform spheroids allows for improved handling and manipulation during transplantation. These findings strongly support the concept that 3D culture systems remain not only a viable option for stem cell culture but also possibly a more attractive alternative to traditional culture techniques to improve the osteogenic potential of human adipose stem cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of monosaccharide sugars on LH-induced differentiation and sugar transport facilitator (SLC2A) expression in sheep theca cells in vitro.

PubMed

Campbell, B K; Kendall, N R; Onions, V; Guo, L; Scaramuzzi, R J

2014-03-01

The aim of the present study was to investigate the effects of glucose, galactose and fructose on the LH-induced differentiation and mRNA expression of sugar transport facilitators (SLC2A) by sheep thecal cells derived from small antral follicles cultured under serum-free conditions for 6 days. The dose and type of monosaccharide had a significant effect on LH-induced androstenedione production by theca cells and there was a significant interaction (P<0.001). Glucose and galactose were used with equal efficiency so that cell numbers and androstenedione production at the end of the culture were comparable. Pharmacological doses of glucose (16.7 mM) inhibited steroidogenesis (P<0.05). Cell numbers and androstenedione production by cells cultured with fructose were lower than for cells cultured with either glucose or galactose (P<0.001). None of the monosaccharides resulted in the production of lactate. Expression of SLC2A1, SLC2A4 and SLC2A8, but not SLC2A5, mRNA was detected in fresh and cultured theca cells. Large doses (16.7 mM) of glucose and fructose, but not galactose, suppressed (P<0.05) SLC2A expression. The results show that glucose and galactose, but not fructose, are readily metabolised via oxidative pathways to support LH-induced differentiation of sheep theca cells. Further work is required to determine the mechanisms resulting in these differences in relation to the established effects of nutrition on reproductive function.

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

PubMed Central

Kean, Thomas J.; Dennis, James E.

2015-01-01

Background Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential. Methods Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically. Results Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions. Conclusions Synoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage. PMID:26075742

Subcloning of three osteoblastic cell lines with distinct differentiation phenotypes from the mouse osteoblastic cell line KS-4.

PubMed

Yamashita, T; Ishii, H; Shimoda, K; Sampath, T K; Katagiri, T; Wada, M; Osawa, T; Suda, T

1996-11-01

Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.

Opposite cytokine synthesis by fibroblasts in contact co-culture with osteosarcoma cells compared with transwell co-cultures.

PubMed

David, Manu S; Kelly, Elizabeth; Zoellner, Hans

2013-04-01

We recently reported exchange of membrane and cytoplasm during contact co-culture between human Gingival Fibroblasts (h-GF) and SAOS-2 osteosarcoma cells, a process we termed 'cellular sipping' to reflect the manner in which cells become morphologically diverse through uptake of material from the opposing cell type, independent of genetic change. Cellular sipping is increased by Tumor Necrosis Factor-α (TNF-α), and we here show for the first time altered cytokine synthesis in contact co-culture supporting cellular sipping compared with co-culture where h-GF and SAOS-2 were separated in transwells. SAOS-2 had often undetectably low cytokine levels, while Interleukin-6 (IL-6), Granulocyte Colony Stimulating Factor (G-CSF) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) were secreted primarily by TNF-α stimulated h-GF and basic Fibroblast Growth Factor (FGF) was prominent in h-GF lysates (p < 0.001). Contact co-cultures permitting cellular sipping had lower IL-6, G-CSF and GM-CSF levels, as well as higher lysate FGF levels compared with TNF-α treated h-GF alone (p < 0.05). The opposite was the case for co-cultures in transwells, with increased IL-6, G-CSF and GM-CSF levels (p < 0.03) and no clear difference in FGF. We thus demonstrate significant phenotypic change in cultures where cellular sipping occurs, potentially contributing to tumor inflammatory responses. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

PubMed

Tagawa, M; Matoba, S; Narita, M; Saito, N; Nagai, T; Imai, K

2008-03-15

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

PubMed

Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

PubMed Central

Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos.

PubMed

Dai, Xiangpeng; Hao, Jie; Zhou, Qi

2009-08-01

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and alphaMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10-30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds.

PubMed

Sellgren, Katelyn L; Ma, Teng

2015-08-01

Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds. © 2014 Wiley Periodicals, Inc.

Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture

PubMed Central

Jacchetti, Emanuela; Emilitri, Elisa; Rodighiero, Simona; Indrieri, Marco; Gianfelice, Antonella; Lenardi, Cristina; Podestà, Alessandro; Ranucci, Elisabetta; Ferruti, Paolo; Milani, Paolo

2008-01-01

Background Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion. Results A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip. Conclusion The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices. PMID:19014710

Reconstruction of Mouse Testicular Cellular Microenvironments in Long-Term Seminiferous Tubule Culture

PubMed Central

Mäkelä, Juho-Antti; Toppari, Jorma; Rivero-Müller, Adolfo; Ventelä, Sami

2014-01-01

Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1) and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA) and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger), LIN28, Gpr125 (G protein-coupled receptor 125), CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs) that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH) which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2–4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to establish in vitro conditions that allow spontaneous reconstruction of testicular cellular microenvironments. PMID:24619130

Modeling human development in 3D culture.

PubMed

Ader, Marius; Tanaka, Elly M

2014-12-01

Recently human embryonic stem cell research has taken on a new dimension - the third dimension. Capitalizing on increasing knowledge on directing pluripotent cells along different lineages, combined with ECM supported three-dimensional culture conditions, it has become possible to generate highly organized tissues of the central nervous system, gut, liver and kidney. Each system has been used to study different aspects of organogenesis and function including physical forces underlying optic cup morphogenesis, the function of disease related genes in progenitor cell control, as well as interaction of the generated tissues with host tissue upon transplantation. Pluripotent stem cell derived organoids represent powerful systems for the study of how cells self-organize to generate tissues with a given shape, pattern and form. Copyright © 2014 Elsevier Ltd. All rights reserved.

Engineering cell aggregates through incorporated polymeric microparticles.

PubMed

Ahrens, Caroline C; Dong, Ziye; Li, Wei

2017-10-15

Ex vivo cell aggregates must overcome significant limitations in the transport of nutrients, drugs, and signaling proteins compared to vascularized native tissue. Further, engineered extracellular environments often fail to sufficiently replicate tethered signaling cues and the complex architecture of native tissue. Co-cultures of cells with microparticles (MPs) is a growing field directed towards overcoming many of these challenges by providing local and controlled presentation of both soluble and tethered proteins and small molecules. Further, co-cultured MPs offer a mechanism to better control aggregate architecture and even to report key characteristics of the local microenvironment such as pH or oxygen levels. Herein, we provide a brief introduction to established and developing strategies for MP production including the choice of MP materials, fabrication techniques, and techniques for incorporating additional functionality. In all cases, we emphasize the specific utility of each approach to form MPs useful for applications in cell aggregate co-culture. We review established techniques to integrate cells and MPs. We highlight those strategies that promote targeted heterogeneity or homogeneity, and we describe approaches to engineer cell-particle and particle-particle interactions that enhance aggregate stability and biological response. Finally, we review advances in key application areas of MP aggregates and future areas of development. Cell-scaled polymer microparticles (MPs) integrated into cellular aggregates have been shown to be a powerful tool to direct cell response. MPs have supported the development of healthy cartilage, islets, nerves, and vasculature by the maintenance of soluble gradients as well as by the local presentation of tethered cues and diffusing proteins and small molecules. MPs integrated with pluripotent stem cells have directed in vivo expansion and differentiation. Looking forward, MPs are expected to support both the characterization and development of in vitro tissue systems for applications such as drug testing platforms. However, useful co-cultures must be designed keeping in mind the limitations and attributes of each material strategy within the context of the overall tissue biology. The present review integrates prospectives from materials development, drug delivery, and tissue engineering to provide a toolbox for the development and application of MPs useful for long-term co-culture within cell aggregates. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

PubMed

Fernando, Elizabeth H; Dicay, Michael; Stahl, Martin; Gordon, Marilyn H; Vegso, Andrew; Baggio, Cristiane; Alston, Laurie; Lopes, Fernando; Baker, Kristi; Hirota, Simon; McKay, Derek M; Vallance, Bruce; MacNaughton, Wallace K

2017-11-01

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner. Copyright © 2017 the American Physiological Society.

Using cell and organ culture models to analyze responses of bone cells to mechanical stimulation.

PubMed

Pitsillides, Andrew A; Rawlinson, Simon C F

2012-01-01

Bone cells of the osteoblastic lineage are responsive to the local mechanical environment. Through integration of a number of possible loading-induced regulatory stimuli, osteocyte, osteoblast, and osteoclast behaviour is organized to fashion a skeletal element of sufficient strength and toughness to resist fracture and crack propagation. Early pre-osteogenic responses had been determined in vivo and this led to the development of bone organ culture models to elucidate other pre-osteogenic responses where osteocytes and osteoblasts retain the natural orientation, connections and attachments to their native extracellular matrix. The application of physiological mechanical loads to bone in these organ culture models generates the regulatory stimuli. As a consequence, these experiments can be used to illustrate the distinctive mechanisms by which osteocytes and osteoblasts respond to mechanical loads and also differences in these responses, suggesting co-ordinated and cooperatively between cell populations. Organ explant cultures are awkward to maintain, and have a limited life, but length of culture times are improving. Monolayer cultures are much easier to maintain and permit the application of a particular mechanical stimulation to be studied in isolation; mainly direct mechanical strain or fluid shear strains. These allow for the response of a single cell type to the applied mechanical stimulation to be monitored precisely.The techniques that can be used to apply mechanical strain to bone and bone cells have not advanced greatly since the first edition. The output from such experiments has, however, increased substantially and their importance is now more broadly accepted. This suggests a growing use of these approaches and an increasing awareness of the importance of the mechanical environment in controlling normal bone cell behaviour. We expand the text to include additions and modifications made to the straining apparatus and update the research cited to support this growing role of cell and organ culture models to analyze responses of bone cells to mechanical stimulation.

Adaptation of Mycobacterium smegmatis to Stationary Phase

PubMed Central

Smeulders, Marjan J.; Keer, Jacquie; Speight, Richard A.; Williams, Huw D.

1999-01-01

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells. PMID:9864340

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

PubMed

Bose, Bipasha; Shenoy P, Sudheer

2016-02-01

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koike, Taro, E-mail: koiket@hirakata.kmu.ac.jp; Wakabayashi, Taketoshi; Mori, Tetsuji

Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown ofmore » Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors. - Highlights: • We established satellite glial cell culture system. • Function of Sox2 in satellite glial cell was examined using siRNA. • Sox2 knockdown downregulated expression level of ErbB2 and ErbB3 mRNA. • Sox2 knockdown increased apoptotic satellite glial cell. • Sox2 promotes satellite glial cell survival through ErbB signaling.« less

Comparative marker analysis after isolation and culture of testicular cells from the immature marmoset.

PubMed

Albert, Silvia; Wistuba, Joachim; Eildermann, Katja; Ehmcke, Jens; Schlatt, Stefan; Gromoll, Joerg; Kossack, Nina

2012-01-01

The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers - anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) - was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis. Copyright © 2012 S. Karger AG, Basel.

Disruption of Cell-Cell Contact-mediated Notch Signaling via Hydrogel Encapsulation Reduces Mesenchymal Stem Cell Chondrogenic Potential

PubMed Central

Chen, Amanda X.; Hoffman, Michael D.; Chen, Caressa S.; Shubin, Andrew D.; Reynolds, Daniel S.; Benoit, Danielle S. W.

2015-01-01

Cell-cell contact-mediated Notch signaling is essential for mesenchymal stem cell (MSC) chondrogenesis during development. However, subsequent deactivation of Notch signaling is also required to allow for stem cell chondrogenic progression. Recent literature has shown that Notch signaling can also influence Wnt/β-catenin signaling, critical for MSC differentiation, through perturbations in cell-cell contacts. Traditionally, abundant cell-cell contacts, consistent with development, are emulated in vitro using pellet cultures for chondrogenesis. However, cells are often encapsulated within biomaterials-based scaffolds, such as hydrogels, to improve therapeutic cell localization in vivo. To explore the role of Notch and Wnt/β-catenin signaling in the context of hydrogel-encapsulated MSC chondrogenesis, we compared signaling and differentiation capacity of MSCs in both hydrogels and traditional pellet cultures. We demonstrate that encapsulation within poly(ethylene glycol) (PEG) hydrogels reduces cell-cell contacts, and both Notch (7.5-fold) and Wnt/β-catenin (84.7-fold) pathway activation. Finally, we demonstrate that following establishment of cell-cell contacts and transient Notch signaling in pellet cultures, followed by Notch signaling deactivation, resulted in a 1.5-fold increase in MSC chondrogenesis. Taken together, these findings support that cellular condensation, and the establishment of initial cell-cell contacts is critical for MSC chondrogenesis, and this process is inhibited by hydrogel encapsulation. PMID:25504509

Ion transport by primary cultures of canine tracheal epithelium: methodology, morphology, and electrophysiology.

PubMed

Welsh, M J

1985-01-01

Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of C1 secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of C1 secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of C1 secretion by airway epithelia.

Development of a functional thyroid model based on an organoid culture system.

PubMed

Saito, Yoshiyuki; Onishi, Nobuyuki; Takami, Hiroshi; Seishima, Ryo; Inoue, Hiroyoshi; Hirata, Yuki; Kameyama, Kaori; Tsuchihashi, Kenji; Sugihara, Eiji; Uchino, Shinya; Ito, Koichi; Kawakubo, Hirofumi; Takeuchi, Hiroya; Kitagawa, Yuko; Saya, Hideyuki; Nagano, Osamu

2018-03-04

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRAS Q61R ) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Continuous production of ethanol from hexoses and pentoses using immobilized mixed cultures of Escherichia coli strains

PubMed Central

Unrean, Pornkamol; Srienc, Friedrich

2010-01-01

We have developed highly efficient ethanologenic E. coli strains that selectively consume pentoses and/or hexoses. Mixed cultures of these strains can be used to selectively adjust the sugar utilization kinetics in ethanol fermentations. Based on the kinetics of sugar utilization, we have designed and implemented an immobilized cell system for the optimized continuous conversion of sugars into ethanol. The results confirm that immobilized mixed cultures support a simultaneous conversion of hexoses and pentoses into ethanol at high yield and at a faster rate than immobilized homogenous cells. Continuous ethanol production has been maintained for several weeks at high productivity with near complete sugar utilization. The control of sugar utilization using immobilized mixed cultures can be adapted to any composition of hexoses and pentoses by adjusting the strain distribution of immobilized cells. The approach, therefore, holds promise for ethanol fermentation from lignocellulosic hydrolysates where the feedstock varies in sugar composition. PMID:20699108

Viral Susceptibility Range of the Fathead Minnow (Pimephales promelas) Poikilothermic Cell Line 1

PubMed Central

Solis, Juan; Mora, Emilio C.

1970-01-01

The viral susceptibility range of a poikilothermic cell line derived from the fathead minnow (Pimephales promelas) (FHM) to infection by a number of homoiothermic viruses representing most of the presently recognized viral groups and a member of the psittacosis-lymphogranuloma-trachoma group of agents was studied. All infectious agents, except poliovirus types 1 and 3, infectious bursal agent, and an avian infectious bronchitis virus (IBV) strain, readily multiplied in the FHM cell culture system, producing a detectable cytopathic effect. Although inconclusive evidence was obtained with two other avian IBV strains, these results indicated the ability of the FHM cell culture system to readily support the propagation of a variety of cytopathogenic homoiothermic viral agents. PMID:5461163

Algal culture studies for CELSS

NASA Technical Reports Server (NTRS)

Radmer, R.; Behrens, P.; Arnett, K.; Gladue, R.; Cox, J.; Lieberman, D.

1987-01-01

Microalgae are well-suited as a component of a Closed Environmental Life Support System (CELSS), since they can couple the closely related functions of food production and atmospheric regeneration. The objective was to provide a basis for predicting the response of CELSS algal cultures, and thus the food supply and air regeneration system, to changes in the culture parameters. Scenedesmus growth was measured as a function of light intensity, and the spectral dependence of light absorption by the algae as well as algal respiration in the light were determined as a function of cell concentration. These results were used to test and confirm a mathematical model that describes the productivity of an algal culture in terms of the competing processes of photosynthesis and respiration. The relationship of algal productivity to cell concentration was determined at different carbon dioxide concentrations, temperatures, and light intensities. The maximum productivity achieved by an air-grown culture was found to be within 10% of the computed maximum productivity, indicating that CO2 was very efficiently removed from the gas stream by the algal culture. Measurements of biomass productivity as a function of cell concentration at different light intensities indicated that both the productivity and efficiency of light utilization were greater at higher light intensities.

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

PubMed

Meller, D; Pires, R T F; Tseng, S C G

2002-04-01

Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

Lipid accumulation by pelletized culture of Mucor circinelloides on corn stover hydrolysate.

PubMed

Reis, Cristiano E R; Zhang, Jianguo; Hu, Bo

2014-09-01

Microbial oil accumulated by fungal cells is a potential feedstock for biodiesel production, and lignocellulosic materials can serve as the carbon source to support the fungal growth. The dilute acid pretreatment of corn stover can effectively break down its lignin structure, and this process generates a hydrolysate containing mostly xylose at very dilute concentration and numerous by-products that may significantly inhibit the cell growth. This study utilized corn stover hydrolysate as the culture media for the growth of Mucor circinelloides. The results showed that Mucor cells formed pellets during the cell growth, which facilitates the cell harvest from dilute solution. The results also showed that the inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), and acetic acid could be avoided if their concentration was low. In fact, all these by-products may be assimilated as carbon sources for the fungal growth. The results proved the feasibility to reuse the cultural broth water for acid pretreatment and then use for subsequent cell cultivation. The results will have a direct impact on the overall water usage of the process.

Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells

PubMed Central

Davami, Fatemeh; Baldi, Lucia; Rajendra, Yashas; M. Wurm, Florian

2014-01-01

The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells. PMID:25317401

Studies on the therapeutic effect of propolis in streptozotocin-induced diabetic mice

NASA Astrophysics Data System (ADS)

Rifa'I, Muhaimin

2017-05-01

Propolis oral administration in diabetic mice can increase the expression of TLR-3 and ameliorate homeostatic imbalance. The TLR-3 expression increased in both B cells and T cells. In this study, we also found that propolis may improve insulin expression in pancreatic beta cells. Administering propolis at a dose of 100-200 mg/mL may significantly increase insulin synthesis. Propolis might protect healthy cells from apoptosis in cisplatin exposure. Cisplatin can induce spleen cells to remain in the G0/G1 phase or to reach the apoptosis stage in the absence of propolis. In contrast, cisplatin, when administered together with propolis to a culture of spleen cells, cannot force the cells to undergo apoptosis. In a culture of spleen cells in the presence of propolis, the cells did not show any responses. This suggests that propolis does not disrupt normal cell physiology and supports cell health when cells are exposed to cisplatin. Furthermore, propolis can suppress the production of the pro-inflammatory cytokine interferon-gamma (IFN-γ).

Emergence of organized structure in co-culture spheroids: Experiments and Theory

NASA Astrophysics Data System (ADS)

Sanford, Roland; Kolbman, Dan; Song, Wei; Wu, Mingming; Ma, Minglin; Das, Moumita

During tissue morphogenesis, from formation of embryos to tumor progression, cells often live and migrate in a heterogeneous environment consisting of many types of cells. To understand how differences in cell mechanobiological properties impact cellular self-organization and migration, we study a co-culture model composed of two distinct cell types confined in a three-dimensional spherical capsule. The cells are modeled as deformable, interacting, self-propelled particles that proliferate at specified timescales. A disordered potential is introduced to mimic the effect of the extracellular matrix (ECM). By varying the mechano-adhesive properties of each type, we investigate how differences in cell stiffness, cell-cell adhesion, and cell-ECM interaction influence collective properties of the binary cell population, such as self-assembly and migration. The predictions of the model are compared to experimental results on co-cutures of breast cancer cells and non-tumorigenic breast epithelial cells. This work was partially supported by a Cottrell College Science Award from the Research Corporation for Science Advancement.

Soft matrix supports osteogenic differentiation of human dental follicle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viale-Bouroncle, Sandra; Voellner, Florian; Moehl, Christoph

Highlights: {yields} Rigid stiffness supports osteogenic differentiation in mesenchymal stem cells (MSCs). {yields} Our study examined stiffness and differentiation of dental follicle cells (DFCs). {yields} Soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs. {yields} DFCs and MSCs react contrarily to soft and rigid surface stiffness. -- Abstract: The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness andmore » cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.« less

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs.

PubMed

Gnedeva, Ksenia; Hudspeth, A J; Segil, Neil

2018-06-01

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Human neuroblastoma (SH-SY5Y) cell culture and differentiation in 3-D collagen hydrogels for cell-based biosensing.

PubMed

Desai, Anu; Kisaalita, William S; Keith, Charles; Wu, Z-Z

2006-02-15

Cell-based three-dimensional systems are desirable in the field of high throughput screening assays due to their potential similarity to in vivo environment. We have used SH-SY5Y human neuroblastoma cells cultured in 3-D collagen hydrogel, confocal microscopy and immunofluorescence staining, to assess the merit of the system as a functional, cell-based biosensor. Our results show differences between 2-D and 3-D resting membrane potential development profile upon differentiation. There was no statistically significant difference in SH-SY5Y proliferation rate between 2-D monolayer and 3-D collagen culture formats. A large percentage of cells (2-D, 91.30% and 3-D, 84.93%) did not develop resting membrane potential value equal to or lower than -40 mV; instead cells exhibited a heterogeneous resting membrane potential distribution. In response to high K(+) (50 mM) depolarization, 3-D cells were less responsive in terms of increase in intracellular Ca(2+), in comparison to 2-D cells, supporting the hypothesis that 2-D cell calcium dynamics may be exaggerated. L-Type Ca(2+) expression levels based on staining results was inconsistent with Bay K 8644 channel activation results, strongly suggesting that either the majority of the channels were non-functional or could not be activated by Bay K 8644. In general, the results in this study confirm the depolarization-induced differences in intracellular calcium release when cultured using a 2-D versus a 3-D matrix.

Direct oxygen supply with polydimethylsiloxane (PDMS) membranes induces a spontaneous organization of thick heterogeneous liver tissues from rat fetal liver cells in vitro.

PubMed

Hamon, Morgan; Hanada, Sanshiro; Fujii, Teruo; Sakai, Yasuyuki

2012-01-01

Oxygen is a vital nutrient for growth and maturation of in vitro cells (e.g., adult hepatocytes). We previously demonstrated that direct oxygenation through a polydimethylsiloxane (PDMS) membrane increases the oxygen supply to cell cultures and improves hepatocyte functions. In this study, we removed limits on oxygen supply to fetal rat liver cells through the use of direct oxygenation through a PDMS membrane to investigate in vitro growth and maturation. We chose fetal liver cells because they are considered a feasible source of liver progenitor cells for regenerative medicine therapy due to their highly efficient maturation and proliferation. Cells from 17-day-old pregnant rats were cultured under 5% and 21% oxygen atmospheres. Some cells were first cultured under 5% oxygen, and then switched to a 21% oxygen atmosphere. When oxygen supply was enhanced by a PDMS membrane, the rat fetal liver cells organized into a complex tissue composed of an epithelium of hepatocytes above a mesenchyme-like tissue. The thickness of this supportive tissue was directly correlated to oxygen concentration and was thicker under 5% oxygen. When cultures were switched from 5% to 21% oxygen, lumen-containing structures were formed in the thick mesenchymal-like tissue and the albumin secretion rate increased. In addition, cells adapted their glycolytic activity to the oxygen concentrations. This system promoted the formation of a functional and organized thick tissue suitable for use in regenerative medicine.

Porcine platelet lysate as a supplement for animal cell culture

PubMed Central

Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

2007-01-01

A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (<10 EU/mL). The porcine platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

Kidney cell electrophoresis, continuing task

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

Simplified Bioreactor For Growing Mammalian Cells

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F.

1995-01-01

Improved bioreactor for growing mammalian cell cultures developed. Designed to support growth of dense volumes of mammalian cells by providing ample, well-distributed flows of nutrient solution with minimal turbulence. Cells relatively delicate and, unlike bacteria, cannot withstand shear forces present in turbulent flows. Bioreactor vessel readily made in larger sizes to accommodate greater cell production quantities. Molding equipment presently used makes cylinders up to 30 centimeters long. Alternative sintered plastic techniques used to vary pore size and quantity, as necessary.

Regulation of cellular function via electromagnetic field frequency and extracellular environment: A theoretical- experimental approach

NASA Astrophysics Data System (ADS)

Taghian, Toloo; Sheikh, Abdul; Narmoneva, Daria; Kogan, Andrei

2015-03-01

Application of external electric field (EF) as a non-pharmacological, non-invasive tool to control cell function is of great therapeutic interest. We developed a theoretical-experimental approach to investigate the biophysical mechanisms of EF interaction with cells in electrode-free physiologically-relevant configuration. Our numerical results demonstrated that EF frequency is the major parameter to control cell response to EF. Non-oscillating or low-frequency EF leads to charge accumulation on the cell surface membrane that may mediate membrane initiated cell responses. In contrast, high-frequency EF penetrates the cell membrane and reaches cell cytoplasm, where it may directly activate intracellular responses. The theoretical predictions were confirmed in our experimental studies of the effects of applied EF on vascular cell function. Results show that non-oscillating EF increases vascular endothelial growth factor (VEGF) expression while field polarity controls cell adhesion rate. High-frequency, but not low frequency, EF provides differential regulation of cytoplasmic focal adhesion kinase and VEGF expression depending on the substrate, with increased expression in cells cultured on RGD-rich synthetic hydrogels, and decreased expression for matrigel culture. The authors acknowledge the financial support from the NSF (DMR-1206784 & DMR-0804199 to AK); the NIH (1R21 DK078814-01A1 to DN) and the University of Cincinnati (Interdisciplinary Faculty Research Support Grant to DN and AK).

Pro-Inflammatory cytokines increases hepatocellular carcinoma cells thermotolerance: Evidence of how local inflammation may negatively impact radiofrequency ablation local control rates

PubMed Central

Douglas, Wade G.; Wang, Yangping; Gibbs, John F.; Tracy, Erin; Kuvshinoff, Boris; Huntoon, Kristin; Baumann, Heinz

2008-01-01

Background Hepatocellular carcinomas (HCC) associated with inflammation that undergo radiofrequency ablation (RFA) appear to have poorer local control rates. Little is known of how mediators of inflammation influence HCC cellular thermotolerance which in part is mediated by heat shock protein 70 (HSP 70). This study determines how inflammatory mediators effect cellular thermotolerance and provides insight into how associated inflammation may impact HCC RFA local control rates. Methods HepG2 cell lines were cultured in control medium (CM) or CM containing conditioned medium of endotoxin-activated macrophage (CMM). Serial dilutions of CMM established microenvironments approximating low, medium and high CMM. All groups underwent a heat shock challenge (HSC) at 45° C for 10 minutes. Western blot, northern blot, densometric analysis, along with Thymidine and clonagenic assays determined how inflammation influenced multiple biologic endpoints. Results Cells cultured in low CMM, expressed significantly more HSP 70 RNA and protein compared to control cells after HSC. The cells also had a higher proliferative and survival rate after HSC compared to control cells. Medium CMM cultured cells had no significant difference in HSP 70 RNA and protein production or proliferation and survival rates after HSC, compared to CM cultured cells. AT high CMM the inhibitory effects of inflammatory mediators prevailed, all the measured endpoints were significantly less compared to CM cultured cells. Conclusions This study demonstrates that inflammation can alter the responsiveness of HCC cells to a HSC in a dose dependent manner. This study supports the clinical observation that HCC associated with chronic inflammation have worse RFA local control rates. PMID:18262552

Platelet-Poor Plasma as a Supplement for Fibroblasts Cultured in Platelet-Rich Fibrin

PubMed Central

Karam, Sarah Arangurem; Noronha, Thaís Gioda; Sartori, Letícia Regina Morello; San Martin, Alissa Schmidt; Demarco, Flávio Fernando; Conde, Marcus Cristian Muniz

2017-01-01

The aim of this study was to evaluate the proliferation and adhesion of mesenchymal cells (3T3/NIH) in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with Platelet-Poor Plasma (PPP) in aPlatelet-Rich Fibrin (PRF) scaffold. Human blood was obtained and processed in a centrifuge considering the equation G=1.12xRx(RPM/1000)2 to obtain PRF and PPP.Cell adhesion and maintenance analyses were performed by MTTassays in a 96 well plate withsupplemented DMEM: PPP (90:10) for 24 hours. Besides, the PRF was deposited in a 48 well plate and 10x104 cells were seeded above each PRF (n=3) with 800µl of DMEM: PPP (90:10) and cultured for 7 days. Histological analysis and the immunohistochemical staining for Vimentin were performed. Results were analyzed by one-way ANOVA in Stata12®. A significant decrease (p<0.05) of cells adhesion in relationship to FBSwas observed. However, a similar ability of cell-maintenance for PPP 10% was observed (P>0.05). Fibroblasts culture for 7 days in PRF supplemented with PPP 10% was possible, showing positive staining for Vimentin. Therefore, PPP cell supplementation decreased the initial adhesion of cells but was able to maintain the proliferation of adhered cells and able to support their viability in PRF.It seems that this method has many clinical advantagessince it provides an autologous and natural scaffold with their respective supplement for cell culture by only one process, without using xenogeneic compounds. This could improve the potential of clinical translational therapies based on the use of PRF cultured cells, promoting the regenerative potential for future use in medicine and dentistry. PMID:28827850

Pooled thrombin-activated platelet-rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts.

PubMed

Tchang, Laurent A; Pippenger, Benjamin E; Todorov, Atanas; Wolf, Francine; Burger, Maximilian G; Jaquiery, Claude; Bieback, Karen; Martin, Ivan; Schaefer, Dirk J; Scherberich, Arnaud

2017-05-01

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose-derived cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Characterization of Fetal Keratinocytes, Showing Enhanced Stem Cell-Like Properties: A Potential Source of Cells for Skin Reconstruction

PubMed Central

Tan, Kenneth K.B.; Salgado, Giorgiana; Connolly, John E.; Chan, Jerry K.Y.; Lane, E. Birgitte

2014-01-01

Summary Epidermal stem cells have been in clinical application as a source of culture-generated grafts. Although applications for such cells are increasing due to aging populations and the greater incidence of diabetes, current keratinocyte grafting technology is limited by immunological barriers and the time needed for culture amplification. We studied the feasibility of using human fetal skin cells for allogeneic transplantation and showed that fetal keratinocytes have faster expansion times, longer telomeres, lower immunogenicity indicators, and greater clonogenicity with more stem cell indicators than adult keratinocytes. The fetal cells did not induce proliferation of T cells in coculture and were able to suppress the proliferation of stimulated T cells. Nevertheless, fetal keratinocytes could stratify normally in vitro. Experimental transplantation of fetal keratinocytes in vivo seeded on an engineered plasma scaffold yielded a well-stratified epidermal architecture and showed stable skin regeneration. These results support the possibility of using fetal skin cells for cell-based therapeutic grafting. PMID:25254345

Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity.

PubMed

Youssef, Hanan H; Hamza, Mervat A; Fayez, Mohamed; Mourad, Elhussein F; Saleh, Mohamed Y; Sarhan, Mohamed S; Suker, Ragab M; Eltahlawy, Asmaa A; Nemr, Rahma A; El-Tahan, Mahmod; Ruppel, Silke; Hegazi, Nabil A

2016-03-01

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >10(6)-10(8) cfu g(-1) were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium.

Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity

PubMed Central

Youssef, Hanan H.; Hamza, Mervat A.; Fayez, Mohamed; Mourad, Elhussein F.; Saleh, Mohamed Y.; Sarhan, Mohamed S.; Suker, Ragab M.; Eltahlawy, Asmaa A.; Nemr, Rahma A.; El-Tahan, Mahmod; Ruppel, Silke; Hegazi, Nabil A.

2015-01-01

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >106–108 cfu g−1 were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium. PMID:26966571

Autophagy contributes to degradation of Hirano bodies

PubMed Central

Kim, Dong-Hwan; Davis, Richard C.; Furukawa, Ruth; Fechheimer, Marcus

2009-01-01

Hirano bodies are actin-rich inclusions reported most frequently in the hippocampus in association with a variety of conditions including neurodegenerative diseases and aging. We have developed a model system for formation of Hirano bodies in Dictyostelium and cultured mammalian cells to permit detailed studies of the dynamics of these structures in living cells. Model Hirano bodies are frequently observed in membrane-enclosed vesicles in mammalian cells consistent with a role of autophagy in the degradation of these structures. Clearance of Hirano bodies by an exocytotic process is supported by images from electron microscopy showing extracellular release of Hirano bodies, and observation of Hirano bodies in the culture medium of Dictyostelium and mammalian cells. An autophagosome marker protein Atg8-GFP was colocalized with model Hirano bodies in wild-type Dictyostelium cells, but not in atg5- or atg1-1 autophagy mutant strains. Induction of model Hirano bodies in Dictyostelium with a high-level expression of 34 kDa ΔEF1 from the inducible discoidin promoter resulted in larger Hirano bodies and a cessation of cell doubling. The degradation of model Hirano bodies still occurred rapidly in autophagy mutant (atg5-) Dictyostelium, suggesting that other mechanisms such as the ubiquitin-mediated proteasome pathway could contribute to the degradation of Hirano bodies. Chemical inhibition of the proteasome pathway with lactacystin significantly decreased the turnover of Hirano bodies in Dictyostelium, providing direct evidence that autophagy and the proteasome can both contribute to degradation of Hirano bodies. Short-term treatment of mammalian cells with either lactacystin or 3-methyl adenine results in higher levels of Hirano bodies and a lower level of viable cells in the cultures, supporting the conclusion that both autophagy and the proteasome contribute to degradation of Hirano bodies. PMID:18989098

3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment.

PubMed

Rebelo, Sofia P; Pinto, Catarina; Martins, Tatiana R; Harrer, Nathalie; Estrada, Marta F; Loza-Alvarez, Pablo; Cabeçadas, José; Alves, Paula M; Gualda, Emilio J; Sommergruber, Wolfgang; Brito, Catarina

2018-05-01

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Galactosylated electrospun membranes for hepatocyte sandwich culture.

PubMed

Chien, Hsiu-Wen; Lai, Juin-Yih; Tsai, Wei-Bor

2014-04-01

In this work, we developed a galactocylated electrospun polyurethane membrane for sandwich culture of hepatocyte sandwich culture. The electrospun fibrous membranes were bio-functionalized with galactose molecules by a UV-crosslinked layer-by-layer polyelectrolyte multilayer deposition technique. The galactosylated electrospun membranes were employed as a top support membrane for the sandwich culture of HepG2/C3A cells on a collagen substrate. Our results demonstrate that HepG2/C3A cells covered by the galactosylated PU membranes form multi-cellular aggregates and lead to improved albumin secretion ability compared to the control membranes (unmodified PU or poly(ethylene imine)-modified PU). Our study reveals the potential of galactosylated electrospun membranes in the application of liver tissue engineering and the regeneration of liver-tissue substitutes. Copyright © 2014 Elsevier B.V. All rights reserved.

Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies.

PubMed

Dinh, Phuong-Uyen C; Cores, Jhon; Hensley, M Taylor; Vandergriff, Adam C; Tang, Junnan; Allen, Tyler A; Caranasos, Thomas G; Adler, Kenneth B; Lobo, Leonard J; Cheng, Ke

2017-06-30

Resident stem and progenitor cells have been identified in the lung over the last decade, but isolation and culture of these cells remains a challenge. Thus, although these lung stem and progenitor cells provide an ideal source for stem-cell based therapy, mesenchymal stem cells (MSCs) remain the most popular cell therapy product for the treatment of lung diseases. Surgical lung biopsies can be the tissue source but such procedures carry a high risk of mortality. In this study we demonstrate that therapeutic lung cells, termed "lung spheroid cells" (LSCs) can be generated from minimally invasive transbronchial lung biopsies using a three-dimensional culture technique. The cells were then characterized by flow cytometry and immunohistochemistry. Angiogenic potential was tested by in-vitro HUVEC tube formation assay. In-vivo bio- distribution of LSCs was examined in athymic nude mice after intravenous delivery. From one lung biopsy, we are able to derive >50 million LSC cells at Passage 2. These cells were characterized by flow cytometry and immunohistochemistry and were shown to represent a mixture of lung stem cells and supporting cells. When introduced systemically into nude mice, LSCs were retained primarily in the lungs for up to 21 days. Here, for the first time, we demonstrated that direct culture and expansion of human lung progenitor cells from pulmonary tissues, acquired through a minimally invasive biopsy, is possible and straightforward with a three-dimensional culture technique. These cells could be utilized in long-term expansion of lung progenitor cells and as part of the development of cell-based therapies for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

Lung cells support osteosarcoma cell migration and survival.

PubMed

Yu, Shibing; Fourman, Mitchell Stephen; Mahjoub, Adel; Mandell, Jonathan Brendan; Crasto, Jared Anthony; Greco, Nicholas Giuseppe; Weiss, Kurt Richard

2017-01-25

Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining. Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

Rational design and application of responsive alpha-helical peptide hydrogels.

PubMed

Banwell, Eleanor F; Abelardo, Edgardo S; Adams, Dave J; Birchall, Martin A; Corrigan, Adam; Donald, Athene M; Kirkland, Mark; Serpell, Louise C; Butler, Michael F; Woolfson, Derek N

2009-07-01

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials. Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely alpha-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of alpha-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.

Rational design and application of responsive α-helical peptide hydrogels

PubMed Central

Banwell, Eleanor F.; Abelardo, Edgardo S.; Adams, Dave J.; Birchall, Martin A.; Corrigan, Adam; Donald, Athene M.; Kirkland, Mark; Serpell, Louise C.; Butler, Michael F.; Woolfson, Derek N.

2009-01-01

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs; and as supports for cell growth and tissue engineering1. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials2-4. Here we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks melt upon heating, whereas those formed via hydrophobic interactions strengthen when warmed. The hSAFs are dual-peptide systems that only gel on mixing, which gives tight control over assembly5. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture. PMID:19543314

Rational design and application of responsive α-helical peptide hydrogels

NASA Astrophysics Data System (ADS)

Banwell, Eleanor F.; Abelardo, Edgardo S.; Adams, Dave J.; Birchall, Martin A.; Corrigan, Adam; Donald, Athene M.; Kirkland, Mark; Serpell, Louise C.; Butler, Michael F.; Woolfson, Derek N.

2009-07-01

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials. Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.

Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats

PubMed Central

Goyal, Ritu; Guvendiren, Murat; Freeman, Onyi; Mao, Yong; Kohn, Joachim

2017-01-01

The design of composite tissue scaffolds containing an extracellular matrix (ECM) and synthetic polymer fibers is a new approach to create bioactive scaffolds that can enhance cell function. Currently, studies investigating the effects of ECM-deposition and decellularization on polymer degradation are still lacking, as are data on optimizing the stability of the ECM-containing composite scaffolds during prolonged cell culture. In this study, we develop fibrous scaffolds using three polymer compositions, representing slow (E0000), medium (E0500), and fast (E1000) degrading materials, to investigate the stability, degradation, and mechanics of the scaffolds during ECM deposition and decellularization, and during the complete cellularization-decell-recell cycle. We report data on percent molecular weight (% Mw) retention of polymeric fiber mats, changes in scaffold stiffness, ECM deposition, and the presence of fibronectin after decellularization. We concluded that the fast degrading E1000 (Mw retention ≤ 50% after 28 days) was not sufficiently stable to allow scaffold handling after 28 days in culture, while the slow degradation of E0000 (Mw retention ≥ 80% in 28 days) did not allow deposited ECM to replace the polymer support. The scaffolds made from medium degrading E0500 (Mw retention about 60% at 28 days) allowed the gradual replacement of the polymer network with cell-derived ECM while maintaining the polymer network support. Thus, polymers with an intermediate rate of degradation, maintaining good scaffold handling properties after 28 days in culture, seem best suited for creating ECM-polymer composite scaffolds. PMID:28085047

Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

PubMed

Sîrbu-Boeţi, Mirela-Patricia; Chivu, Mihaela; Pâslaru, Liliana Livia; Efrimescu, C; Herlea, V; Pecheanu, C; Moldovan, Lucia; Dragomir, Laura; Bleotu, Coralia; Ciucur, Elena; Vidulescu, Cristina; Vasilescu, Mihaela; Boicea, Anişoara; Mănoiu, S; Ionescu, M I; Popescu, I

2009-01-01

Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.

Rhodobacter capsulatus gains a competitive advantage from respiratory nitrate reduction during light-dark transitions.

PubMed

Ellington, M J K; Richardson, D J; Ferguson, S J

2003-04-01

Rhodobacter capsulatus N22DNAR(+) possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR(+) during multiple light-dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR(+). After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR(+) culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light-dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR(+)) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light-dark cycles, such that in mixed culture N22DNAR(+) outcompetes N22. Exposure to light-dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.

A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

PubMed

Ahmadian Baghbaderani, Behnam; Tian, Xinghui; Scotty Cadet, Jean; Shah, Kevan; Walde, Amy; Tran, Huan; Kovarcik, Don Paul; Clarke, Diana; Fellner, Thomas

2016-01-01

Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

Micropatterns of Matrigel for three-dimensional epithelial cultures.

PubMed

Sodunke, Temitope R; Turner, Keneshia K; Caldwell, Sarah A; McBride, Kevin W; Reginato, Mauricio J; Noh, Hongseok Moses

2007-09-01

Three-dimensional (3D) epithelial culture models are widely used to promote a physiologically relevant microenvironment for the study of normal and aberrant epithelial organization. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hindered by the lack of existing platforms for large-scale 3D cellular studies. Current 3D standard culture does not allow for single spheroid or 'acinus' analysis required for high-throughput systems. Here, we present general strategies for creating bulk micropatterns of Matrigel that can be used as a platform for 3D epithelial culture and cell-based assays at the single acinus level. Both buried and free-standing micropatterns of Matrigel were created using modified soft lithography techniques such as microtransfer molding (microTM) and dry lift-off technique. Surface modification of poly(dimethylsiloxane) (PDMS) with oxygen plasma followed by treatment with poly(2-hydroxy-ethylmethacrylate) (poly-HEMA) was sufficient to promote deformation-free release of Matrigel patterns. In addition, a novel dual-layer dry lift-off technique was developed to simultaneously generate patterns of Matrigel and poly-HEMA on a single substrate. We also demonstrate that the micropatterned Matrigel can support 3D culture originating from a single normal human mammary epithelial (MCF-10A) cell or a human breast cancer cell (MDA-MB-231) with comparable phenotypes to standard 3D culture techniques. Culture of normal MCF-10A cells on micropatterned Matrigel resulted in formation of structures with the characteristic apoptosis of centrally located cells and formation of hollow lumens. Moreover, the carcinoma cell line showed their characteristic formation of disorganized invasive cellular clusters, lacking the normal epithelial architecture on micropatterned Matrigel. Hence, micropatterned Matrigel can be used as a 3D epithelial cell-based platform for a wide variety of applications in epithelial and cancer biology, tissue engineering, as well as gene/drug screening technology.

Assessment of the Tumorigenic Potential of Spontaneously Immortalized and hTERT-Immortalized Cultured Dental Pulp Stem Cells

PubMed Central

Wilson, Ryan; Urraca, Nora; Skobowiat, Cezary; Hope, Kevin A.; Miravalle, Leticia; Chamberlin, Reed; Donaldson, Martin; Seagroves, Tiffany N.

2015-01-01

Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest-derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell-derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage-independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)-immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony-forming potential in soft agar for the spontaneously immortalized and hTERT-immortalized DPSC lines relative to low-passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT-immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases. Significance This study demonstrated that immortalized dental pulp stem cells (DPSCs) do not form tumors in animals and that immortalized DPSCs can be differentiated into neurons in culture. These results lend support to the use of primary and immortalized DPSCs for future therapeutic approaches to treatment of neurobiological diseases. PMID:26032749

Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

PubMed

Sorkio, Anni; Porter, Patrick J; Juuti-Uusitalo, Kati; Meenan, Brian J; Skottman, Heli; Burke, George A

2015-09-01

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, I.S.; Gaa, S.T.; Rogers, T.B.

The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G{sub i}) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effectsmore » of both ANG II and carbachol, suggesting a role for G{sub i} in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated {sup 32}P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G{sub i} is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G{sub i} is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G{sub i} in heart.« less

Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer invasion

PubMed Central

Konen, J.; Summerbell, E.; Dwivedi, B.; Galior, K.; Hou, Y.; Rusnak, L.; Chen, A.; Saltz, J.; Zhou, W.; Boise, L. H.; Vertino, P.; Cooper, L.; Salaita, K.; Kowalski, J.; Marcus, A. I.

2017-01-01

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape. PMID:28497793

Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver.

PubMed

Suzuki, A; Zheng, Y; Kondo, R; Kusakabe, M; Takada, Y; Fukao, K; Nakauchi, H; Taniguchi, H

2000-12-01

Stem cells responsible for tissue maintenance and repair are found in a number of organs. However, hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. To address this issue, we set up a culture system in which primitive hepatic progenitor cells formed colonies. By combining this culture system with fluorescence-activated cell sorting (FACS), cells forming colonies containing distinct hepatocytes and cholangiocytes were identified in the fetal mouse liver. These cells express both CD49f and CD29 (alpha6 and beta1 integrin subunits), but do not mark for hematopoietic antigens such as CD45, TER119, and c-Kit. When transplanted into the spleen, these cells migrated to the recipient liver and differentiated into liver parenchymal cells. Our data demonstrate that hepatic progenitor cells are enriched by FACS and suggest approaches to supplanting organ allografting and improving artificial-organ hepatic support.

Melatonin and its precursors in Y79 human retinoblastoma cells: Effect of sodium butyrate

NASA Technical Reports Server (NTRS)

Deng, Mei Hua; Coviella, Ignacio Lopez G.; Lynch, Harry J.; Wurtman, Richard J.

1991-01-01

The release of melatonin and the production of its precursors, S-hydroxytryptophan and serotonin, in cultured Y79 human retinoblastoma cells were studied. This biosynthetic capability was found to be dependent on cell differentiation, which was initiated by culturing Y79 cells for 7 days in dishes coated with poly-D-lysine to promote cell adhesion to the surface of the culture dishes. Differentiation was further induced by exposing the cell monolayer to sodium butyrate (3 mM) for 3 days. This protocol dramatically increased the release of melatonin, and the syntheses of 5-hydroxytryptophan and serotonin in response to forskolin stimulation. Exposure to dopamine (10 micro-M) or L-DOPA (100 micro-M) markedly diminished the forskolin-stimulated release of melatonin, as well as the production of 5-hydroxytryptophan and serotonin. These observations indicate that Y79 cells represent a primitive cell line which, following appropriate differentiation (e.g. treatment with sodium butyrate) can display biochemical characteristics similar to those of the human retina. Moreover, serotonin synthesis and melatonin release appear to be coupled in Y79 cells. The inhibition of melatonin release by dopamine supports the hypothesis that in these cells, melatonin and dopamine are components of a retinal feedback loop.

GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

PubMed Central

Krtolica, Ana; Larocque, Nick; Genbacev, Olga; Ilic, Dusko; Coppe, Jean-Philippe; Patil, Christopher K.; Zdravkovic, Tamara; McMaster, Michael; Campisi, Judith; Fisher, Susan J.

2012-01-01

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ~6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical–basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical–basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation—βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell–cell contacts can trigger adoption of a neuronal fate. PMID:21396766

Silver sub-nanoclusters electrocatalyze ethanol oxidation and provide protection against ethanol toxicity in cultured mammalian cells.

PubMed

Selva, Javier; Martínez, Susana E; Buceta, David; Rodríguez-Vázquez, María J; Blanco, M Carmen; López-Quintela, M Arturo; Egea, Gustavo

2010-05-26

Silver atomic quantum clusters (AgAQCs), with two or three silver atoms, show electrocatalytic activities that are not found in nanoparticles or in bulk silver. AgAQCs supported on glassy carbon electrodes oxidize ethanol and other alcohols in macroscopic electrochemical cells in acidic and basic media. This electrocatalysis occurs at very low potentials (from approximately +200 mV vs RHE), at physiological pH, and at ethanol concentrations that are found in alcoholic patients. When mammalian cells are co-exposed to ethanol and AgAQCs, alcohol-induced alterations such as rounded cell morphology, disorganization of the actin cytoskeleton, and activation of caspase-3 are all prevented. This cytoprotective effect of AgAQCs is also observed in primary cultures of newborn rat astrocytes exposed to ethanol, which is a cellular model of fetal alcohol syndrome. AgAQCs oxidize ethanol from the culture medium only when ethanol and AgAQCs are added to cells simultaneously, which suggests that cytoprotection by AgAQCs is provided by the ethanol electro-oxidation mediated by the combined action of AgAQCs and cells. Overall, these findings not only show that AgAQCs are efficient electrocatalysts at physiological pH and prevent ethanol toxicity in cultured mammalian cells, but also suggest that AgAQCs could be used to modify redox reactions and in this way promote or inhibit biological reactions.

Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.

PubMed

Makoolati, Zohreh; Movahedin, Mansoureh; Forouzandeh-Moghadam, Mehdi

2016-12-01

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs. © 2016 The Author(s).

Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens

PubMed Central

March, Sandra; Ramanan, Vyas; Trehan, Kartik; Ng, Shengyong; Galstian, Ani; Gural, Nil; Scull, Margaret A.; Shlomai, Amir; Mota, Maria; Fleming, Heather E.; Khetani, Salman R.; Rice, Charles M.; Bhatia, Sangeeta N.

2018-01-01

Studying human hepatotropic pathogens such as hepatitis B and C viruses and malaria will be necessary for understanding host-pathogen interactions, and developing therapy and prophylaxis. Unfortunately, existing in vitro liver models typically employ either cell lines that exhibit aberrant physiology, or primary human hepatocytes in culture configurations wherein they rapidly lose their hepatic functional phenotype. Stable, robust, and reliable in vitro primary human hepatocyte models are needed as platforms for infectious disease applications. For this purpose, we describe the application of micropatterned co-cultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive cells. Using this system, we demonstrate how to recapitulate in vitro liver infection by the hepatitis B and C viruses and Plasmodium pathogens. In turn, the MPCC platform can be used to uncover aspects of host-pathogen interactions, and has the potential to be used for medium-throughput drug screening and vaccine development. PMID:26584444

Algal culture studies related to a closed ecological life support system

NASA Technical Reports Server (NTRS)

Radmer, R.; Behrens, P.; Fernandez, E.; Ollinger, O.; Howell, C.

1984-01-01

Studies on the steady-state long-term (4 month) culture of Scenedesmus obliquus algae, maintained in an annular air-lift column operated as a turbidostat, were carried out to evaluate the life-supporting possibilities of this system. Chlorophyll production and cell number as functions of the dry weight were linear at constant illumination. Productivity (measured as the product of dry weight, mg/ml, and the growth rate, ml/hr) vs. dry weight rose linearly until the cell density reached a level at which light became limiting (89 percent absorption of the photosynthetically active radiation). In the initial, linear portion of the curve, the productivity was limited by cell growth at the given light intensity. The maximum dilution rate of the system corresponded to the doubling time of 13.4 hr, about half the maximum rate, with a productivity of 80 percent of the maximum theoretical productivity. The high light utilization efficiencies were contributed by the low (10 percent of full sunlight) incident intensities.

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

PubMed Central

Hao, Weidong; Bernard, Katie; Patel, Nita; Ulbrandt, Nancy; Feng, Hui; Svabek, Catherine; Wilson, Susan; Stracener, Christina; Wang, Kathy; Suzich, JoAnn; Blair, Wade

2012-01-01

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors. PMID:23035218

Tailoring microfluidic systems for organ-like cell culture applications using multiphysics simulations

NASA Astrophysics Data System (ADS)

Hagmeyer, Britta; Schütte, Julia; Böttger, Jan; Gebhardt, Rolf; Stelzle, Martin

2013-03-01

Replacing animal testing with in vitro cocultures of human cells is a long-term goal in pre-clinical drug tests used to gain reliable insight into drug-induced cell toxicity. However, current state-of-the-art 2D or 3D cell cultures aiming at mimicking human organs in vitro still lack organ-like morphology and perfusion and thus organ-like functions. To this end, microfluidic systems enable construction of cell culture devices which can be designed to more closely resemble the smallest functional unit of organs. Multiphysics simulations represent a powerful tool to study the various relevant physical phenomena and their impact on functionality inside microfluidic structures. This is particularly useful as it allows for assessment of system functions already during the design stage prior to actual chip fabrication. In the HepaChip®, dielectrophoretic forces are used to assemble human hepatocytes and human endothelial cells in liver sinusoid-like structures. Numerical simulations of flow distribution, shear stress, electrical fields and heat dissipation inside the cell assembly chambers as well as surface wetting and surface tension effects during filling of the microchannel network supported the design of this human-liver-on-chip microfluidic system for cell culture applications. Based on the device design resulting thereof, a prototype chip was injection-moulded in COP (cyclic olefin polymer). Functional hepatocyte and endothelial cell cocultures were established inside the HepaChip® showing excellent metabolic and secretory performance.

Regulation of Cellular Calcium in Vestibular Supporting Cells by Otopetrin 1

PubMed Central

Kim, Euysoo; Hyrc, Krzysztof L.; Speck, Judith; Lundberg, Yunxia W.; Salles, Felipe T.; Kachar, Bechara; Goldberg, Mark P.; Warchol, Mark E.

2010-01-01

Otopetrin 1 (OTOP1) is a multitransmembrane domain protein, which is essential for mineralization of otoconia, the calcium carbonate biominerals required for vestibular function, and the normal sensation of gravity. The mechanism driving mineralization of otoconia is poorly understood, but it has been proposed that supporting cells and a mechanism to maintain high concentrations of calcium are critical. Using Otop1 knockout mice and a utricular epithelial organ culture system, we show that OTOP1 is expressed at the apex of supporting cells and functions to increase cytosolic calcium in response to purinergic agonists, such as adenosine 5′-triphosphate (ATP). This is achieved by blocking mobilization of calcium from intracellular stores in an extracellular calcium-dependent manner and by mediating influx of extracellular calcium. These data support a model in which OTOP1 acts as a sensor of the extracellular calcium concentration near supporting cells and responds to ATP in the endolymph to increase intracellular calcium levels during otoconia mineralization. PMID:20554841

Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers.

PubMed

Levato, Riccardo; Visser, Jetze; Planell, Josep A; Engel, Elisabeth; Malda, Jos; Mateos-Timoneda, Miguel A

2014-09-01

Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

Novel anisotropic engineered cardiac tissues: studies of electrical propagation.

PubMed

Bursac, Nenad; Loo, Yihua; Leong, Kam; Tung, Leslie

2007-10-05

The goal of this study was to engineer cardiac tissue constructs with uniformly anisotropic architecture, and to evaluate their electrical function using multi-site optical mapping of cell membrane potentials. Anisotropic polymer scaffolds made by leaching of aligned sucrose templates were seeded with neonatal rat cardiac cells and cultured in rotating bioreactors for 6-14 days. Cells aligned and interconnected inside the scaffolds and when stimulated by a point electrode, supported macroscopically continuous, anisotropic impulse propagation. By culture day 14, the ratio of conduction velocities along vs. across cardiac fibers reached a value of 2, similar to that in native neonatal ventricles, while action potential duration and maximum capture rate, respectively, decreased to 120ms and increased to approximately 5Hz. The shorter culture time and larger scaffold thickness were associated with increased incidence of sustained reentrant arrhythmias. In summary, this study is the first successful attempt to engineer a cm(2)-size, functional anisotropic cardiac tissue patch.

Hybrid Thin Film Organosilica Sol-Gel Coatings To Support Neuronal Growth and Limit Astrocyte Growth.

PubMed

Capeletti, Larissa Brentano; Cardoso, Mateus Borba; Dos Santos, João Henrique Zimnoch; He, Wei

2016-10-07

Thin films of silica prepared by a sol-gel process are becoming a feasible coating option for surface modification of implantable neural sensors without imposing adverse effects on the devices' electrical properties. In order to advance the application of such silica-based coatings in the context of neural interfacing, the characteristics of silica sol-gel are further tailored to gain active control of interactions between cells and the coating materials. By incorporating various readily available organotrialkoxysilanes carrying distinct organic functional groups during the sol-gel process, a library of hybrid organosilica coatings is developed and investigated. In vitro neural cultures using PC12 cells and primary cortical neurons both reveal that, among these different types of hybrid organosilica, the introduction of aminopropyl groups drastically transforms the silica into robust neural permissive substrate, supporting neuron adhesion and neurite outgrowth. Moreover, when this organosilica is cultured with astrocytes, a key type of glial cells responsible for glial scar response toward neural implants, such cell growth promoting effect is not observed. These findings highlight the potential of organo-group-bearing silica sol-gel to function as advanced coating materials to selectively modulate cell response and promote neural integration with implantable sensing devices.

Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells

PubMed Central

Lukiw, Walter J.; Pogue, Aileen I.

2007-01-01

Iron- and aluminum-sulfate together, at nanomolar concentrations, trigger the production of reactive oxygen species (ROS) in cultures of human brain cells. Previous studies have shown that following ROS induction, a family of pathogenic brain genes that promote inflammatory signalling, cellular apoptosis and brain cell death is significantly over-expressed. Notably, iron- and aluminum-sulfate induce genes in cultured human brain cells that exhibit expression patterns similar to those observed to be up-regulated in moderate- to late-stage Alzheimer's disease (AD). In this study we have extended our investigations to analyze the expression of micro RNA (miRNA) populations in iron- and aluminum-sulfate treated human neural cells in primary culture. The main finding was that these ROS-generating neurotoxic metal sulfates also up-regulate a specific set of miRNAs that includes miR-9, miR-125b and miR-128. Notably, these same miRNAs are up-regulated in AD brain. These findings further support the idea that iron- and aluminum-sulfates induce genotoxicity via a ROS-mediated up-regulation of specific regulatory elements and pathogenic genes that redirect brain cell fate towards progressive dysfunction and apoptotic cell death. PMID:17629564

Methods for the preparation of an autologous serum-free cultured epidermis and for autografting applications.

PubMed

Wille, John J; Burdge, Jeremy J; Park, Jong Y

2014-01-01

Cell culture techniques for producing a three-dimensional autologous epidermal autograft (cultured epidermal autograft) suitable for tissue grafting and wound healing procedures are described. This chapter commences with surgical biopsy of patient's skin tissue, further reduction of skin tissues to keratinocyte cells by enzymatic treatment, and recovery of viable adult keratinocytes in a new balanced buffered salt media supportive of the growth of clonally enriched isolated basal keratinocytes. Culture techniques required for the formation of a hole-free monolayer of undifferentiated basal keratinocytes without the use of an organotypic matrix substrate are accomplished with a specially designed nutrient basal media (HECK 109) that is a chemically defined and subsequent culture in this serum-free culture media supplemented with hormones and two human recombinant protein growth factors (EGF and IGF-1). Further culture techniques and media manipulations, including brief exposure to β-TGF to induce reversible G1-phase growth arrest, are followed by para-synchronous induction of a multilayered stratification and keratinizing epidermal differentiation, yielding a living three-dimensional epidermis formed entirely in cell culture. Protocols are listed for its enzymatic removal, floatation, and transfer for shipment to the clinic ready for surgical grafting to the self-same patient's debrided chronic leg ulcers. Recent clinical trial results have demonstrated the utility and efficacy of these grafts in forming durably healed chronic wounds.

Oxygen consumption in T-47D cells immobilized in alginate.

PubMed

Larsen, B E; Sandvik, J A; Karlsen, J; Pettersen, E O; Melvik, J E

2013-08-01

Encapsulation or entrapment of cells is increasingly being used in a wide variety of scientific studies for tissue engineering and development of novel medical devices. The effect on cell metabolism of such systems is, in general, not well characterized. In this work, a simple system for monitoring respiration of cells embedded in 3-D alginate cultures was characterized. T-47D cells were cultured in alginate gels. Oxygen concentration curves were recorded within cell-gel constructs using two different sensor systems, and cell viability and metabolic state were characterized using confocal microscopy and commercially available stains. At sufficient depth within constructs, recorded oxygen concentration curves were not significantly influenced by influx of oxygen through cell-gel layers and oxygen consumption rate could be calculated simply by dividing oxygen loss in the system per time, by the number of cells. This conclusion was supported by a 3-D numeric simulation. For the T-47D cells, the oxygen consumption rate was found to be 61 ± 6 fmol/cell/h, 3-4 times less than has previously been found for these cells, when grown exponentially in monolayer culture. The experimental set-up presented here may be varied in multiple ways by changing the cell-gel construct 3-D microenvironment, easily allowing investigation of a variety of factors on cell respiration. © 2013 John Wiley & Sons Ltd.

Accelerated production of antigen-specific T-cells for pre-clinical and clinical applications using Gas-permeable Rapid Expansion cultureware (G-Rex)

PubMed Central

Vera, Juan F.; Brenner, Lara J.; Gerdemann, Ulrike; Ngo, Minhtran C.; Sili, Uluhan; Liu, Hao; Wilson, John; Dotti, Gianpietro; Heslop, Helen E.; Leen, Ann M.; Rooney, Cliona M.

2009-01-01

The clinical manufacture of antigen-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy is limited by the complexity and time required to produce large numbers with the desired function and specificity. The culture conditions required are rigorous, and in some cases only achieved in 2cm2 wells in which cell growth is limited by gas exchange, nutrients and waste accumulation. Bioreactors developed to overcome these issues tend to be complex, expensive and not always conducive to CTL growth. We observed that antigen-specific CTL undergo seven to ten divisions post-stimulation. However the expected CTL numbers were achieved only in the first week of culture. By recreating the culture conditions present during this first week - low frequency of antigen-specific T-cells and high frequency of feeder cells - we were able to increase CTL expansion to expected levels which could be sustained for several weeks without affecting phenotype or function. However, the number of 24-well plates needed was excessive and cultures required frequent media changes, increasing complexity and manufacturing costs. Therefore, we evaluated novel gas-permeable culture devices (G-Rex) with a silicone membrane at the base allowing gas exchange to occur uninhibited by depth of medium above. This system effectively supports the expansion of CTL and actually increases output by up to 20-fold while decreasing required technician time. Importantly, this amplified cell expansion is not due to more cell divisions but to reduced cell death. This bioprocess optimization increased T-cell output while decreasing the complexity and cost of CTL manufacture, making cell therapy more accessible. PMID:20445351

A Study of Parameters Affecting Fibroblast Morphology in Response to an Applied Mechanical Force

NASA Technical Reports Server (NTRS)

Grymes, Rosalind A.; Sawyer, Christine

1994-01-01

A precisely controlled stretch/relaxation regimen (20% elongation at 6.6 cycles/min) was applied to normal human fetal, neonatal and aged dermal fibroblasts cultured on flexible membranes. Culture conditions included poly (NH2) or collagen type I coated substrate membranes; control cultures were grown on the same pliable material in the absence of applied stretch. Direct observation and immunofluorescence analyses revealed a progressive change in cell body orientation limited to the stretched dermal fibroblast cultures. Monolayers gradually (over 4 days) acquired a symmetric, radial distribution equivalent to the biaxial array of the applied force. At high seeding density, alignment was inhibited in the fetal cell cultures. This cell strain required collagen type I coating for optimal attachment to the flexible membrane, preferring growth in three-dimensional cell 'balls' on the poly(NH2) coated substrate. Neonatal cells also required the collagen type I coating, but both neonatal and aged dermal fibroblasts aligned efficiently at all seeding densities examined. The randomly oriented neonatal cells on the unstretched control membranes spontaneously detached at confluence, as a single cell sheet. Their aligned counterparts did not detach until the applied stretch stimulus was removed. Low concentrations of cytochalasin D (62.5 ng/ml) disrupted the stretch-related alignment response. Rhodamine phalloidin staining visualized fewer actin stress fibers in stretched, aligned cells than in controls. Both intercellular interactions and cytoskeletal integrity mediate the response to mechanical strain. Normal rabbit corneal stroma fibroblasts (NRC) were also analyzed, and failed to orient under these conditions. This cell type may require a different regimen, or a longer time period, to demonstrate alignment behavior. Supported by NASA Space Biology RTOP 199-40-22 and the NASA-ARC Director's Discretionary Fund.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

PubMed

Arias, María E; Ross, Pablo J; Felmer, Ricardo N

2013-01-01

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

IONIC EFFECTS ON LIGNIFICATION AND PEROXIDASE IN TISSUE CULTURES

PubMed Central

Lipetz, Jacques; Garro, Anthony J.

1965-01-01

Crown-gall tumor tissue cultures release peroxidase into the medium in response to the concentration of specific ions in the medium. This release is not due to diffusion from cut surfaces or injured cells. Calcium, magnesium, and ammonium were, in that order, most effective in increasing peroxidase release. The enzyme was demonstrated cytochemically on the cell walls and in the cytoplasm. Cell wall fractions, exhaustively washed in buffer, still contained bound peroxidase. This bound peroxidase could be released by treating the wall fractions with certain divalent cations or ammonium. The order of effectiveness for removing the enzyme from the washed cell walls is: Ca++ ≈ Sr++ > Ba++ > Mg++ > NH4 +. These data support the thesis presented that specific ions can control the deposition of lignin on cell walls by affecting the peroxidase levels on these walls. PMID:19866650

Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

PubMed

Campeau, Jody L; Wu, Gengshu; Bell, John R; Rasmussen, Jay; Sim, Valerie L

2013-01-01

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures.

PubMed

Azghadi, Seyed Mohammad Reza; Suciu, Maria; Gruia, Alexandra Teodora; Barbu-Tudoran, Lucian; Cristea, Mirabela Iustina; Mic, Ani Aurora; Muntean, Danina; Nica, Dragos Vasile; Mic, Felix Aurel

2016-08-01

The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4-CD8- double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells' influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes.

Microphysiological systems meet hiPSC technology - New tools for disease modeling of liver infections in basic research and drug development.

PubMed

Raasch, Martin; Fritsche, Enrico; Kurtz, Andreas; Bauer, Michael; Mosig, Alexander S

2018-06-14

Complex cell culture models such as microphysiological models (MPS) mimicking human liver functionality in vitro are in the spotlight as alternative to conventional cell culture and animal models. Promising techniques like microfluidic cell culture or micropatterning by 3D bioprinting are gaining increasing importance for the development of MPS to address the needs for more predictivity and cost efficiency. In this context, human induced pluripotent stem cells (hiPSCs) offer new perspectives for the development of advanced liver-on-chip systems by recreating an in vivo like microenvironment that supports the reliable differentiation of hiPSCs to hepatocyte-like cells (HLC). In this review we will summarize current protocols of HLC generation and highlight recently established MPS suitable to resemble physiological hepatocyte function in vitro. In addition, we are discussing potential applications of liver MPS for disease modeling related to systemic or direct liver infections and the use of MPS in testing of new drug candidates. Copyright © 2018. Published by Elsevier B.V.

Heterogeneity of Clonal Expansion and Maturation-Linked Mutation Acquisition in Hematopoietic Progenitors in Human Acute Myeloid Leukemia

PubMed Central

Walter, Roland B.; Laszlo, George S.; Lionberger, Jack M.; Pollard, Jessica A.; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Othus, Megan; Rafii, Shahin; Meshinchi, Soheil; Appelbaum, Frederick R.; Bernstein, Irwin D.

2014-01-01

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML) but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition, and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34+/CD33− cells revealed polyclonal growth in highly curable AMLs, suggesting mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34+/CD33− cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications. PMID:24721792

Inhibition of caspases prevents ototoxic and ongoing hair cell death

NASA Technical Reports Server (NTRS)

Matsui, Jonathan I.; Ogilvie, Judith M.; Warchol, Mark E.

2002-01-01

Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

Determination of the Elastic Moduli of a Single Cell Cultured on a Rigid Support by Force Microscopy.

PubMed

Garcia, Pablo D; Garcia, Ricardo

2018-06-19

The elastic response of a living cell is affected by its physiological state. This property provides mechanical fingerprints of a cell's dysfunctionality. The softness (kilopascal range) and thickness (2-15 μm) of mammalian cells imply that the force exerted by the probe might be affected by the stiffness of the solid support. This observation makes infinite sample thickness models unsuitable to describe quantitatively the forces and deformations on a cell. Here, we report a general theory to determine the true Young's moduli of a single cell from a force-indentation curve. Analytical expressions are deduced for common geometries such as flat punches, paraboloids, cones, needles, and nanowires. For a given cell and indentation, the influence of the solid support on the measurements is reduced by using sharp and high aspect ratio tips. The theory is validated by finite element simulations. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

PubMed

Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

2015-04-01

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

PubMed

Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

2014-07-01

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2.

PubMed Central

Small, F J; Ensign, S A

1995-01-01

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone. PMID:7592382

Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed.

PubMed

Chen, Allen Kuan-Liang; Chew, Yi Kong; Tan, Hong Yu; Reuveny, Shaul; Weng Oh, Steve Kah

2015-02-01

Large amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. MSCs were expanded in stirred cultures on Cytodex 3 microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. Minimum essential medium-α-based medium supported faster MSC growth on microcarriers than did Dulbecco's modified Eagle's medium (doubling time, 31.6 ± 1.4 vs 42 ± 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 × 10(6) cells/mL with confluent cell concentration of 4.7 × 10(4)cells/cm(2) was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/10(6)cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/10(6)cells. We have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

PubMed

Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

2018-03-05

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A comparison study on the behavior of human endometrial stem cell-derived osteoblast cells on PLGA/HA nanocomposite scaffolds fabricated by electrospinning and freeze-drying methods.

PubMed

Namini, Mojdeh Salehi; Bayat, Neda; Tajerian, Roxana; Ebrahimi-Barough, Somayeh; Azami, Mahmoud; Irani, Shiva; Jangjoo, Saranaz; Shirian, Sadegh; Ai, Jafar

2018-03-27

An engineered tissue structure is an artificial scaffold combined with cells and signaling factors. Among various polymers, the polylactide-co-glycolide/hydroxyapatite (PLGA/HA) has attracted much attention due to their optimal properties. The aim of this study was to study the behavior of human endometrial stem cell (hEnSC)-derived osteoblast cells cultured on PLGA/HA nanocomposite scaffolds. hEnSCs were isolated and exposed to osteogenic media for 21 days. Differentiated cells were cultured on PLGA/HA synthetic scaffolds. The PLGA/HA-based nanocomposite scaffolds were fabricated using either electrospinning or freeze-drying methods. Behavior of the cells was evaluated a week after seeding hEnSC-derived osteoblast-like cells on these scaffolds. Osteogenesis was investigated in terms of alkaline phosphatase activity, gene expression, immunocytochemistry (ICC), proliferation, and scanning electron microscopy (SEM). Moreover, scaffold properties, such as pore size and morphology of the cells, onto the scaffolds were evaluated using SEM. Furthermore, biocompatibility of these scaffolds was confirmed by 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The matrix mineralization was proved by alizarin red staining, and the osteogenic media-treated cultures positively expressed osteocalcin and osteopontin markers. Moreover, qRT-PCR results confirmed the positive gene expression of osteopontin and osteonectin in the differentiated osteoblast-like cells. The results of behavior assessment of the cultured cells on electrospinning and freeze-dried scaffolds showed that the behavior of the cultured cells on the freeze-dried PLGA/HA scaffolds was significantly better than the electrospinning PLGA/HA scaffolds. It has been shown that the freeze-dried PLGA/HA nanocomposite scaffolds can appropriately support the attachment and proliferation of the differentiated osteoblast cells and are a suitable candidate for bone tissue engineering.

Chlamydia trahomatis in cell culture. I. Comparison of efficiencies of infection in several chemically defined media, at various pH and temperature values, and after exposure to diethylaminoethyl-dextran

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rota, T.R.; Nichols, R.L.

1973-10-01

Three chemically defined cell culture media, Eagle minimum essential medium (MEM) with Earle basal salt solution, Eagle MEM with Hanks basal salt solution, and a modified Eagle MEM, were tested and found capable of supporting the development of Chlamydia trachomatis in /sup 60/Co-treated McCoy cells. The enhancement of trachoma infection by diethylaminoethyl-dextran (DEAE-D) was greater at pH values closer to neutrality than at and other pH values measured at the start of the experiments. Centrifugation of the trachoma inoculum onto cell monolayers at 33 C increased the number of inclusions when compared to centrifugation at 20 C. When the inoculummore » was centrifuged onto cell monolayers and subsequent incubation was at temperatures ranging from 34 to 39 C, the greatest number of inclusions was observed after incubation from 35 through 37 C. Enhancement of the trachoma infection by DEAE-D was tested at temperatures ranging from 35 to 37 C. These cultures had three- to fivefold increases in inclusions when compared to previously reported experiments in which DEAE-D- treated cultures were incubated at 34 C. (auth)« less

In Vitro Endothelialization of Biodegradable Vascular Grafts Via Endothelial Progenitor Cell Seeding and Maturation in a Tubular Perfusion System Bioreactor.

PubMed

Melchiorri, Anthony J; Bracaglia, Laura G; Kimerer, Lucas K; Hibino, Narutoshi; Fisher, John P

2016-07-01

A critical challenge to the success of biodegradable vascular grafts is the establishment of a healthy endothelium. To establish this monolayer of endothelial cells (ECs), a variety of techniques have been developed, including cell seeding. Vascular grafts may be seeded with relevant cell types and allowed to mature before implantation. Due to the low proliferative ability of adult ECs and issues with donor site morbidity, there has been increasing interest in using endothelial progenitor cells (EPCs) for vascular healing procedures. In this work, we combined the proliferative and differentiation capabilities of a commercial cell line of early EPCs with an established bioreactor system to support the maturation of cell-seeded vascular grafts. All components of the vascular graft and bioreactor setup are commercially available and allow for complete customization of the scaffold and culturing system. This bioreactor setup enables the control of flow through the graft, imparting fluid shear stress on EPCs and affecting cellular proliferation and differentiation. Grafts cultured with EPCs in the bioreactor system demonstrated greatly increased cell populations and neotissue formation compared with grafts seeded and cultured in a static system. Increased expression of markers for mature endothelial tissues were also observed in bioreactor-cultured EPC-seeded grafts. These findings suggest the distinct advantages of a customizable bioreactor setup for the proliferation and maturation of EPCs. Such a strategy may be beneficial for utilizing EPCs in vascular tissue engineering applications.

Three-Dimensional Cell Behavior in Microgels

NASA Astrophysics Data System (ADS)

Bhattacharjee, Tapomoy; Palmer, Glyn; Ghivizzani, Steven; Keselowsky, Benjamin; Sawyer, W. Gregory; Angelini, Thomas

The number of dimensions in which particles can freely move strongly influences the collective behavior that emerges from their individual fluctuations. Thus, in 2D systems of cells in petri-dishes, our growing understanding of collective migration may be insufficient to explain cell behavior in 3D tissues. To study cell behavior in 3D, polymer scaffolds are used. Contemporary designs of 3D cell growth scaffolds enable cell migration and proliferative expansion by incorporating of degradable motifs. Matrix degradation creates space for cells to move and proliferate. However, different cell types and experimental conditions require the design of different scaffolds to optimize degradation with specific cell behaviors. By contrast, liquid like solids made from packed microgels can yield under cell generated stresses, allowing for cell motion without the need for scaffold degradation. Moreover, the use of microgels as 3D culture media allows arranging cells in arbitrary structures, harvesting cells, and delivering drugs and nutrients. Preliminary data describing cell behavior in 3D microgel culture will be presented. This material is based on work supported by the National Science Foundation under Grant No. DMR-1352043.

Production and in vitro evaluation of soy protein-based biofilms as a support for human keratinocyte and fibroblast culture.

PubMed

Curt, Sèverine; Subirade, Muriel; Rouabhia, Mahmoud

2009-06-01

This study presents results on soy protein isolate (SPI) biofilm production and the corresponding effect on the stability and toxicity of the derived films. SPI biofilms were prepared from SPI chemically treated with formaldehyde at various concentrations (0%, 1%, 2%, and 3%) as cross-linking agents. In vitro SPI biofilm degradation was evaluated as a function of water absorption leading to weight and size modifications. SPI biofilm toxicity was determined as a function of human keratinocyte and fibroblast adhesion, viability, and proliferation. Cytokine gene expression supported this using reverse transcriptase polymerase chain reaction techniques. Our results confirm that SPI can be used to produce biofilms. The resulting SPI biofilms without formaldehyde swell significantly, which leads to their physical instability. Formaldehyde treatment enhanced the mechanical properties of these biofilms by covalently cross-linking polypeptide chains. The decreased water absorption was dependent on the amount of formaldehyde present. SPI biofilms with 2% and 3% formaldehyde were highly stable and easier to manipulate than those with 0% and 1% formaldehyde. Tissue culture analyses revealed that the SPI biofilms without formaldehyde were non-toxic to human cells (keratinocytes and fibroblasts). The presence of formaldehyde in biofilms did not have any effects on cell viability, adhesion, or proliferation. This was supported by the high level of messenger RNA expression of interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha by the keratinocytes and of IL-6 and IL-8 by the fibroblasts. Overall, we produced a stable, non-toxic soy protein support, which may be of potential interest in medical applications such as cell culture matrices and damaged tissue replacement.

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.

PubMed

Müller, I; Kordowich, S; Holzwarth, C; Spano, C; Isensee, G; Staiber, A; Viebahn, S; Gieseke, F; Langer, H; Gawaz, M P; Horwitz, E M; Conte, P; Handgretinger, R; Dominici, M

2006-01-01

Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Evaluation of organic anion-transporting polypeptide 1B1 and CYP3A4 activities in primary human hepatocytes and HepaRG cells cultured in a dynamic three-dimensional bioreactor system.

PubMed

Ulvestad, Maria; Darnell, Malin; Molden, Espen; Ellis, Ewa; Åsberg, Anders; Andersson, Tommy B

2012-10-01

The long-term stability of liver cell functions is a major challenge when studying hepatic drug transport, metabolism, and toxicity in vitro. The aim of the present study was to investigate organic anion-transporting polypeptide (OATP) 1B1 and CYP3A4 activities in fresh primary human hepatocytes and differentiated cryopreserved HepaRG cells when cultured in a three-dimensional (3D) bioreactor system. OATP1B1 activity was determined by loss from media experiments of [(3)H]estradiol-17β-D-glucuronide and atorvastatin acid (ATA) for up to 7 days in culture. ATA metabolite formation was determined at days 3 to 4 to evaluate CYP3A4 activity. Overall, the results showed that freshly isolated human hepatocytes inoculated in the bioreactor retained OATP1B1 activity for at least 7 days, whereas in HepaRG cells no OATP1B1 activity was observed beyond day 2. The activity data were in agreement with immunohistochemical stainings, which showed that OATP1B1 protein expression was preserved for at least 9 days in fresh human hepatocytes, whereas OATP1B1 was expressed markedly lower in HepaRG cells after 9 days in culture. Fresh human hepatocytes and HepaRG cells exhibited similar CYP3A4 activity in bioreactor culture, and immunohistochemical stainings supported these findings. Activity and mRNA expression of OATP1B1 and CYP3A4 in primary human hepatocytes compared with HepaRG cells in fresh suspensions were in agreement with data obtained in bioreactor culture. In conclusion, freshly isolated human hepatocytes cultured in a 3D bioreactor system preserve both OATP1B1 and CYP3A4 activities, allowing long-term in vitro studies on drug disposition and toxicity.

Long-term culture of human liver tissue with advanced hepatic functions.

PubMed

Ng, Soon Seng; Xiong, Anming; Nguyen, Khanh; Masek, Marilyn; No, Da Yoon; Elazar, Menashe; Shteyer, Eyal; Winters, Mark A; Voedisch, Amy; Shaw, Kate; Rashid, Sheikh Tamir; Frank, Curtis W; Cho, Nam Joon; Glenn, Jeffrey S

2017-06-02

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

Formation of industrial mixed culture biofilm in chlorophenol cultivated medium of microbial fuel cell

NASA Astrophysics Data System (ADS)

Hassan, Huzairy; Jin, Bo; Dai, Sheng; Ngau, Cornelius

2016-11-01

The formation of microbial biofilm while maintaining the electricity output is a challenging topic in microbial fuel cell (MFC) studies. This MFC critical factor becomes more significant when handling with industrial wastewater which normally contains refractory and toxic compounds. This study explores the formation of industrial mixed culture biofilm in chlorophenol cultivated medium through observing and characterizing microscopically its establishment on MFC anode surface. The mixed culture was found to develop its biofilm on the anode surface in the chlorophenol environment and established its maturity and dispersal stages with concurrent electricity generation and phenolic degradation. The mixed culture biofilm engaged the electron transfer roles in MFC by generating current density of 1.4 mA/m2 and removing 53 % of 2,4-dichlorophenol. The results support further research especially on hazardous wastewater treatment using a benign and sustainable method.

A modular suite of hardware enabling spaceflight cell culture research

NASA Technical Reports Server (NTRS)

Hoehn, Alexander; Klaus, David M.; Stodieck, Louis S.

2004-01-01

BioServe Space Technologies, a NASA Research Partnership Center (RPC), has developed and operated various middeck payloads launched on 23 shuttle missions since 1991 in support of commercial space biotechnology projects. Modular cell culture systems are contained within the Commercial Generic Bioprocessing Apparatus (CGBA) suite of flight-qualified hardware, compatible with Space Shuttle, SPACEHAB, Spacelab and International Space Station (ISS) EXPRESS Rack interfaces. As part of the CGBA family, the Isothermal Containment Module (ICM) incubator provides thermal control, data acquisition and experiment manipulation capabilities, including accelerometer launch detection for automated activation and thermal profiling for culture incubation and sample preservation. The ICM can accommodate up to 8 individually controlled temperature zones. Command and telemetry capabilities allow real-time downlink of data and video permitting remote payload operation and ground control synchronization. Individual cell culture experiments can be accommodated in a variety of devices ranging from 'microgravity test tubes' or standard 100 mm Petri dishes, to complex, fed-batch bioreactors with automated culture feeding, waste removal and multiple sample draws. Up to 3 levels of containment can be achieved for chemical fixative addition, and passive gas exchange can be provided through hydrophobic membranes. Many additional options exist for designing customized hardware depending on specific science requirements.

Ultrathin Transparent Membranes for Cellular Barrier and Co-Culture Models

PubMed Central

Carter, Robert N.; Casillo, Stephanie M.; Mazzocchi, Andrea R.; DesOrmeaux, Jon-Paul S.; Roussie, James A.; Gaborski, Thomas R.

2017-01-01

Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100–300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives. PMID:28140345

The Herpes Simplex Virus Type 1 Latency-Associated Transcript Can Protect Neuron-Derived C1300 and Neuro2A Cells from Granzyme B-Induced Apoptosis and CD8 T-Cell Killing▿

PubMed Central

Jiang, Xianzhi; Alami Chentoufi, Aziz; Hsiang, Chinhui; Carpenter, Dale; Osorio, Nelson; BenMohamed, Lbachir; Fraser, Nigel W.; Jones, Clinton; Wechsler, Steven L.

2011-01-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo. PMID:21177822

Defining an optimal surface chemistry for pluripotent stem cell culture in 2D and 3D

NASA Astrophysics Data System (ADS)

Zonca, Michael R., Jr.

Surface chemistry is critical for growing pluripotent stem cells in an undifferentiated state. There is great potential to engineer the surface chemistry at the nanoscale level to regulate stem cell adhesion. However, the challenge is to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a high-throughput polymerization and screening platform, a chemically defined, synthetic polymer grafted coating that supports strong attachment and high expansion capacity of pluripotent stem cells has been discovered using mouse embryonic stem (ES) cells as a model system. This optimal substrate, N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA) that is grafted on 2D synthetic poly(ether sulfone) (PES) membrane, sustains the self-renewal of ES cells (up to 7 passages). DMAPMA supports cell attachment of ES cells through integrin beta1 in a RGD-independent manner and is similar to another recently reported polymer surface. Next, DMAPMA has been able to be transferred to 3D by grafting to synthetic, polymeric, PES fibrous matrices through both photo-induced and plasma-induced polymerization. These 3D modified fibers exhibited higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D PES membranes. Our results indicated that desirable surfaces in 2D can be scaled to 3D and that both surface chemistry and structural dimension strongly influence the growth and differentiation of pluripotent stem cells. Lastly, the feasibility of incorporating DMAPMA into a widely used natural polymer, alginate, has been tested. Novel adhesive alginate hydrogels have been successfully synthesized by either direct polymerization of DMAPMA and methacrylic acid blended with alginate, or photo-induced DMAPMA polymerization on alginate nanofibrous hydrogels. In particular, DMAPMA-coated alginate hydrogels support strong ES cell attachment, exhibiting a concentration dependency of DMAPMA. This research provides a new avenue for stem cell culture and maintenance using an optimal organic-based chemistry.

Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

PubMed Central

Shakibaei, Mehdi; Buhrmann, Constanze; Kraehe, Patricia; Shayan, Parviz; Lueders, Cora; Goel, Ajay

2014-01-01

Objective Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. Methods High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Results Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Conclusion Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract). PMID:24404205

Curcumin chemosensitizes 5-fluorouracil resistant MMR-deficient human colon cancer cells in high density cultures.

PubMed

Shakibaei, Mehdi; Buhrmann, Constanze; Kraehe, Patricia; Shayan, Parviz; Lueders, Cora; Goel, Ajay

2014-01-01

Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50-60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).

Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

PubMed

Verginelli, Fabio; Perconti, Silvia; Vespa, Simone; Schiavi, Francesca; Prasad, Sampath Chandra; Lanuti, Paola; Cama, Alessandro; Tramontana, Lorenzo; Esposito, Diana Liberata; Guarnieri, Simone; Sheu, Artenca; Pantalone, Mattia Russel; Florio, Rosalba; Morgano, Annalisa; Rossi, Cosmo; Bologna, Giuseppina; Marchisio, Marco; D'Argenio, Andrea; Taschin, Elisa; Visone, Rosa; Opocher, Giuseppe; Veronese, Angelo; Paties, Carlo T; Rajasekhar, Vinagolu K; Söderberg-Nauclér, Cecilia; Sanna, Mario; Lotti, Lavinia Vittoria; Mariani-Costantini, Renato

2018-05-01

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

Directing Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Impact of Medium Composition and Culture Condition.

PubMed

Petry, L; Kippenberger, S; Meissner, M; Kleemann, J; Kaufmann, R; Rieger, U M; Wellenbrock, S; Reichenbach, G; Zöller, N; Valesky, E

2018-04-28

Adipose-derived stem cells (ASC) are known to transdifferentiate into a wide range of different cell species in vitro including along the epidermal lineage. This property makes them a promising tool for regenerative medicine in order to restore the epidermal barrier. The present study is dedicated to identify in vitro conditions enabling transdifferentiation to a keratinocyte-like phenotype. Especially, the impact of different culture conditions (media compositions, 2D-, 3D-cultures) and extracellular matrix (ECM) molecules was evaluated. ASC derived from subcutaneous abdominal fat were characterized by stemness associated markers and subjected to different media. Epithelial differentiation in 2D cultures was monitored by pan-cytokeratin expression using flow cytometry and immunocytochemistry. In order to evaluate the impact of different ECM molecules on epidermal stratification, 3D cultures were produced, lifted to the air-liquid-interface (ALI) and examined by histological analysis and quantitative real-time RT-PCR. We identified a medium composition containing retinoic acid, hydrocortisone, ascorbic acid and BMP-4 enabling maximum pan-cytokeratin expression in 2D cultures. Moreover, adhesion to type IV collagen further promotes the pan-cytokeratin expression. When cultures were lifted to the ALI, significant stratification was observed, particularly in supports coated with type IV collagen or fibronectin. Moreover, epidermal differentiation markers (involucrin, cytokeratin 1 and 14) become induced. Conditions with hampered wound healing such as non-healing ulcers demand new treatment regimes. The here introduced optimized protocols for transdifferentiation of ASC into keratinocyte-like cells may help to establish more effective treatment procedures. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics.

PubMed

Schuurman, W; Harimulyo, E B; Gawlitta, D; Woodfield, T B F; Dhert, W J A; van Weeren, P R; Malda, J

2016-04-01

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of osteoblastic culture conditions on the structure of poly(DL-lactic-co-glycolic acid) foam scaffolds

NASA Technical Reports Server (NTRS)

Goldstein, A. S.; Zhu, G.; Morris, G. E.; Meszlenyi, R. K.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

1999-01-01

Poly(DL-lactic-co-glycolic acid) (PLGA) foams are an osteoconductive support that holds promise for the development of bone tissue in vitro and implantation into orthopedic defects. Because it is desirable that foams maintain their shape and size, we examined a variety of foams cultured in vitro with osteoblastic cells. Foams were prepared with different porosities and pore sizes by the method of solvent casting/porogen leaching using 80, 85, and 90 wt% NaCl sieved with particle sizes of 150-300 and 300-500 microm and characterized by mercury intrusion porosimetry. Foams seeded with cells were found to have volumes after 7 days in static culture that decreased with increasing porosity: the least porous exhibited no change in volume while the most porous foams decreased by 39 +/- 10%. In addition, a correlation was observed between decreasing foam volume after 7 days in culture and decreasing internal surface area of the foams prior to seeding. Furthermore, foams prepared with the 300-500 microm porogen had lower porosities, greater mean wall thicknesses between adjacent pores, and larger volumes after 7 days in culture than those prepared with the smaller porogen. Two culture conditions for maintaining cells, static and agitated (in a rotary vessel), were found to have similar influences on foam size, cell density, and osteoblastic function for 7 and 14 days in culture. Finally, we examined unseeded foams in aqueous solutions of pH 3.0, 5.0, and 7.4 and found no significant decrease in foam size with degradation. This study demonstrates that adherent osteoblastic cells may collapse very porous PLGA foams prepared by solvent casting/particulate leaching: a potentially undesirable property for repair of orthopedic defects.

Effect of Prevascularization on In Vivo Vascularization of Poly(Propylene fumarate)/Fibrin Scaffolds

PubMed Central

Mishra, Ruchi; Roux, Brianna M.; Posukonis, Megan; Bodamer, Emily; Brey, Eric M.; Fisher, John P.; Dean, David

2016-01-01

The importance of vascularization in the field of bone tissue engineering has been established by previous studies. The present work proposes a novel poly(propylene fumarate) (PPF)/fibrin composite scaffold for the development of vascularized neobone tissue. The effect of prevascularization (i.e., in vitro pre-culture prior to implantation) with human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) on in vivo vascularization of scaffolds was determined. Five conditions were studied: no pre-culture (NP), 1 week preculture (1P), 2 week pre-culture (2P), 3 week pre-culture (3P), and scaffolds without cells (control, C). Scaffolds were implanted subcutaneously in a severe combined immunodeficiency (SCID) mice model for 9 days. During in vitro studies, CD31 staining showed a significant increase in vascular network area over 3 weeks of culture. Vascular density was significantly higher in vivo when comparing NP to 3P groups. Immunohistochemical staining of human CD-31 expression indicated spreading of vascular networks with increasing pre-culture time. These vascular networks were perfused with mouse blood indicated by perfused lectin staining in human CD-31 positive vessels. Our results demonstrate that in vitro prevascularization supports in vivo vascularization in PPF/fibrin scaffolds. PMID:26606451

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells.

PubMed

Dong, Haodi; Tang, Ya-Jie; Ohashi, Ryo; Hamel, Jean-François P

2005-01-01

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.

Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

PubMed

Russell, K A; Koch, T G

2016-03-01

Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

Combining microbial cultures for efficient production of electricity from butyrate in a microbial electrochemical cell.

PubMed

Miceli, Joseph F; Garcia-Peña, Ines; Parameswaran, Prathap; Torres, César I; Krajmalnik-Brown, Rosa

2014-10-01

Butyrate is an important product of anaerobic fermentation; however, it is not directly used by characterized strains of the highly efficient anode respiring bacteria (ARB) Geobacter sulfurreducens in microbial electrochemical cells. By combining a butyrate-oxidizing community with a Geobacter rich culture, we generated a microbial community which outperformed many naturally derived communities found in the literature for current production from butyrate and rivaled the highest performing natural cultures in terms of current density (∼ 11A/m(2)) and Coulombic efficiency (∼ 70%). Microbial community analyses support the shift in the microbial community from one lacking efficient ARB in the marine hydrothermal vent community to a community consisting of ∼ 80% Geobacter in the anode biofilm. This demonstrates the successful production and adaptation of a novel microbial culture for generating electrical current from butyrate with high current density and high Coulombic efficiency, by combining two mixed microbial cultures containing complementing biochemical pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

2009-02-06

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPAR{alpha}) signaling. Furthermore,more » using PPAR{alpha} agonists and antagonists, we also analyzed the effect of PPAR{alpha} signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.« less

Xylogenesis in zinnia (Zinnia elegans) cell cultures: unravelling the regulatory steps in a complex developmental programmed cell death event.

PubMed

Iakimova, Elena T; Woltering, Ernst J

2017-04-01

Physiological and molecular studies support the view that xylogenesis can largely be determined as a specific form of vacuolar programmed cell death (PCD). The studies in xylogenic zinnia cell culture have led to many breakthroughs in xylogenesis research and provided a background for investigations in other experimental models in vitro and in planta . This review discusses the most essential earlier and recent findings on the regulation of xylem elements differentiation and PCD in zinnia and other xylogenic systems. Xylogenesis (the formation of water conducting vascular tissue) is a paradigm of plant developmental PCD. The xylem vessels are composed of fused tracheary elements (TEs)-dead, hollow cells with patterned lignified secondary cell walls. They result from the differentiation of the procambium and cambium cells and undergo cell death to become functional post-mortem. The TE differentiation proceeds through a well-coordinated sequence of events in which differentiation and the programmed cellular demise are intimately connected. For years a classical experimental model for studies on xylogenesis was the xylogenic zinnia (Zinnia elegans) cell culture derived from leaf mesophyll cells that, upon induction by cytokinin and auxin, transdifferentiate into TEs. This cell system has been proven very efficient for investigations on the regulatory components of xylem differentiation which has led to many discoveries on the mechanisms of xylogenesis. The knowledge gained from this system has potentiated studies in other xylogenic cultures in vitro and in planta. The present review summarises the previous and latest findings on the hormonal and biochemical signalling, metabolic pathways and molecular and gene determinants underlying the regulation of xylem vessels differentiation in zinnia cell culture. Highlighted are breakthroughs achieved through the use of xylogenic systems from other species and newly introduced tools and analytical approaches to study the processes. The mutual dependence between PCD signalling and the differentiation cascade in the program of TE development is discussed.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

PubMed

Sierralta, Walter D; Epuñan, María J; Reyes, José M; Valladares, Luis E; Pino, Ana M

2008-01-01

A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.

Enhancing methyl parathion degradation by the immobilization of Burkholderia sp. isolated from agricultural soils.

PubMed

Fernández-López, Maikel Gilberto; Popoca-Ursino, Carolina; Sánchez-Salinas, Enrique; Tinoco-Valencia, Raunel; Folch-Mallol, Jorge Luis; Dantán-González, Edgar; Laura Ortiz-Hernández, Ma

2017-10-01

Organophosphate pesticides are of great interest for research because they are currently the most commonly used pesticides. In this study, a bacterial strain capable of completely degrading methyl parathion (MP) was isolated from agricultural soils in central Mexico. This strain was designated strain S5-2 and was identified as Burkholderia cenocepacia. To increase degradation yields, cells were immobilized on three different supports: powdered zeolite and Opuntia sp. and Agave sp. fibers. The results indicated a significant increase in MP hydrolysis and p-nitrophenol (PNP) degradation with immobilized cells compared to free cell cultures. Furthermore, immobilized cells were capable of withstanding and degrading higher concentrations of PNP compared to cell suspension cultures. The cell viability in the free cell cultures, as well as PNP degradation, was affected at concentrations greater than 25 mg/L. In contrast, cells immobilized on Opuntia sp. and Agave sp. fibers completely degraded PNP at concentrations of 100 mg/L. To verify that MP solution toxicity was decreased by B. cenocepacia strain S5-2 via pesticide degradation, we measured the acetylcholinesterase activity, both before and after treatment with bacteria. The results demonstrate that the activity of acetylcholinesterase was unaffected after MP degradation by bacteria. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells

PubMed Central

Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra

2016-01-01

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2. PMID:27413259

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

PubMed

Miranda, Dante; Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra; Montoya, Margarita

2016-01-01

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

PubMed Central

Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

2013-01-01

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche.

PubMed

Tan, Jing; Liu, Ting; Hou, Li; Meng, Wentong; Wang, Yuchun; Zhi, Wei; Deng, Li

2010-10-01

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a "biomimetic niche." The CD34(+) cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2-5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34(+) cells from umbilical cord blood in the 3D system increased 3.3-4.8 folds when compared with the initial CD34(+) cells. CD34(+)/CD38(-) cells accounted for 82-90% of CD34(+) cells. After 5 weeks, CD34(+)/CD38(-) cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10(3) vs. 1.0 ± 0.5 × 10(4), p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10(3) vs. 2.5 ± 0.7 × 10(2), p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6-9.3 folds vs. 1.0-1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2-7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.

Application of Quality by Design to the characterization of the cell culture process of an Fc-Fusion protein.

PubMed

Rouiller, Yolande; Solacroup, Thomas; Deparis, Véronique; Barbafieri, Marco; Gleixner, Ralf; Broly, Hervé; Eon-Duval, Alex

2012-06-01

The production bioreactor step of an Fc-Fusion protein manufacturing cell culture process was characterized following Quality by Design principles. Using scientific knowledge derived from the literature and process knowledge gathered during development studies and manufacturing to support clinical trials, potential critical and key process parameters with a possible impact on product quality and process performance, respectively, were determined during a risk assessment exercise. The identified process parameters were evaluated using a design of experiment approach. The regression models generated from the data allowed characterizing the impact of the identified process parameters on quality attributes. The main parameters having an impact on product titer were pH and dissolved oxygen, while those having the highest impact on process- and product-related impurities and variants were pH and culture duration. The models derived from characterization studies were used to define the cell culture process design space. The design space limits were set in such a way as to ensure that the drug substance material would consistently have the desired quality. Copyright © 2012 Elsevier B.V. All rights reserved.

An analysis of the productivity of a CELSS continuous algal culture system

NASA Technical Reports Server (NTRS)

Radmer, R.; Behrens, P.; Fernandez, E.; Arnett, K.

1986-01-01

One of the most attractive aspects of using algal cultures as plant components for a Closed Ecological Life Support Systems (CELSS) is the efficiency with which they can be grown. Although algae are not necessarily intrinsically more efficient than higher plants, the ease which they can be handled and manipulated (more like chemical reagents than plants), and the culturing techniques available, result in much higher growth rates than are usually attainable with higher plants. Furthermore, preliminary experiments have demonstrated that algal growth and physiology is not detectable altered in a microgravity environment, (1) whereas the response of higher plants to zero gravity is unknown. In order to rationally design and operate culture systems, it is necessary to understand how the macroparameters of a culture system, e.g., productivity, are related to the physiological aspects of the algal culture. A first principles analysis of culture system is discussed, and a mathematical model that describes the relationship of culture productivity to the cell concentration of light-limited culture is derived. The predicted productivity vs cell concentration curve agrees well with the experimental data obtained to test this model, indicating that this model permits an accurate prediction of culture productivity given the growth parameters of the system.

Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

NASA Technical Reports Server (NTRS)

Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

1997-01-01

Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis.

PubMed

Reimonn, Thomas M; Park, Seo-Young; Agarabi, Cyrus D; Brorson, Kurt A; Yoon, Seongkyu

2016-09-01

Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016. © 2016 American Institute of Chemical Engineers.

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mobasseri, Rezvan; Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National University of Singapore, 117576; Tian, Lingling

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on differentmore » substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation. - Highlights: • Bioactive molecules modified surface is a strategy to design biomimicry scaffold. • Bi-functional Tat-derived peptide (R-pept) enhanced MSCs adhesion and proliferation. • R-pept showed similar influences to fibronectin on FA formation and attachment.« less

Generic Raman-based calibration models enabling real-time monitoring of cell culture bioreactors.

PubMed

Mehdizadeh, Hamidreza; Lauri, David; Karry, Krizia M; Moshgbar, Mojgan; Procopio-Melino, Renee; Drapeau, Denis

2015-01-01

Raman-based multivariate calibration models have been developed for real-time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO-based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration. © 2015 American Institute of Chemical Engineers.

Morphology of isolated mouse inner cell masses developing in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiley, L.M.; Spindle, A.I.; Pedersen, R.A.

1978-01-01

The purpose of this study was to examine the developmental capacity of the mouse inner cell mass (ICM) in the absence of the trophoblast. ICMs were isolated from blastocysts by immunosurgery and cultured under conditions that support egg cylinder formation by intact blastocysts. After 2 or 3 days of culture, the ICMs consisted of an outer layer of endoderm and an inner layer of ectoderm that had cavitated centrally. By 4 or 5 days of culture, 25 to 60% of these ICMs had developed into paired cysts, apparently by secondary cavity formation. The inner cell layer surrounding this secondary cavitymore » resembled the extraembryonic ectoderm of cultured egg cylinders. By 6 days of culture, 60% of the ICMs had expanded into yolk sac-like structures that subsequently produced capillaries containing blood cells. The ICMs appeared to develop mesoderm in two distinct ways. A few of them developed mesoderm as a third layer of cells in the cleft separating endoderm and ectoderm, presumably by migrating from the inner, ectodermal layer, through the primitive streak, as in the intact egg cylinder. In the rest of the ICMs the embryonic ectoderm gradually differentiated into mesoderm while still in the inner layer, without primitive streak formation. We suggest, therefore, that the continuous presence of the trophoblast or of its derivatives is not required for the cytodifferentiation of mesoderm although it may be important in establishing embryonic polarity or in providinginductive signals necessary for the morphogenetic aspects of mesoderm differentiation, specifically primitive streak formation.« less