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Sample records for 2-d cell culture

  1. Proton NMR characterization of intact primary and metastatic melanoma cells in 2D & 3D cultures.

    PubMed

    Ramachandran, Gokula Krishnan; Yeow, Chen Hua

    2017-03-16

    To characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures. Primary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures. These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed. In monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells. In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids. This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines. Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures. This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo. This study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor. Differences in the cell culture method does plays an important role in phospholipid composition of the cells.

  2. Modeling Physiological Events in 2D vs. 3D Cell Culture.

    PubMed

    Duval, Kayla; Grover, Hannah; Han, Li-Hsin; Mou, Yongchao; Pegoraro, Adrian F; Fredberg, Jeffery; Chen, Zi

    2017-07-01

    Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research. Copyright © 2017 the American Physiological Society.

  3. Modeling Physiological Events in 2D vs. 3D Cell Culture

    PubMed Central

    Duval, Kayla; Grover, Hannah; Han, Li-Hsin; Mou, Yongchao; Pegoraro, Adrian F.; Fredberg, Jeffery

    2017-01-01

    Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research. PMID:28615311

  4. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models.

    PubMed

    Yue, Xiaoshan; Lukowski, Jessica K; Weaver, Eric M; Skube, Susan B; Hummon, Amanda B

    2016-12-02

    Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

  5. In vitro evaluation of curcumin effects on breast adenocarcinoma 2D and 3D cell cultures.

    PubMed

    Abuelba, Hussam; Cotrutz, Carmen Elena; Stoica, Bogdan Alexandru; Stoica, Laura; Olinici, DoiniŢa; Petreuş, Tudor

    2015-01-01

    Breast adenocarcinoma cell line MDA-MB-231, even if it expresses low levels of E-cadherin, still readily form multicellular aggregates of cells, namely spheroids. Curcumin is a diarylheptanoid antitumoral drug while it significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. Curcumin photoactivation may enhance antiapoptotic role against cancer cells. To evaluate the effect of low curcumin concentrations, ranged from 1.9 to 15 μM, with and without photoactivation, using a manufactured 670 nm LED-matrix. A secondary aim was to evaluate the ideal method to produce easy-to-use tumor cell spheroids, comparing two low adherence plate supports. Breast adenocarcinoma cell line MDA-MB-231 were cultured according to 2D monolayer and 3D spheroid models then submitted to normal and photoactivated curcumin in micromolar concentrations. MTT assay was used to evaluate cell viability following curcumin application on cells. On 2D cell cultures, curcumin inhibits cell tumor development and proliferation at concentrations of 15 μM, with a viability of 65.7% at 48 hours incubation time. A decreased viability up to 25% for a concentration of 15 μM was recorded following photoactivation and cytotoxic action on breast cancer tumor cell line continued at concentrations of 7.5 and 3.75 μM. Curcumin photoactivation increases pro-apoptotic effects in both 2D and 3D tumor cell culture models and also responsiveness to curcumin is slightly reduced in spheroid-like structures. Thus, 3D tumor cell culture systems appear to be the ideal environment for in vitro assays regarding anticancer drug effects on cell viability.

  6. Bridging the gap: from 2D cell culture to 3D microengineered extracellular matrices

    PubMed Central

    Li, Yanfen

    2016-01-01

    Historically the culture of mammalian cells in the laboratory has been performed on planar substrates with media cocktails that are optimized to maintain phenotype. However, it is becoming increasingly clear that much of biology discerned from 2D studies does not translate well to the 3D microenvironment. Over the last several decades, 2D and 3D microengineering approaches have been developed that better recapitulate the complex architecture and properties of in vivo tissue. Inspired by the infrastructure of the microelectronics industry, lithographic patterning approaches have taken center stage because of the ease in which cell-sized features can be engineered on surfaces and within a broad range of biocompatible materials. Patterning and templating techniques enable precise control over extracellular matrix properties including: composition, mechanics, geometry, cell-cell contact, and diffusion. In this review article we will explore how the field of engineered extracellular matrices has evolved with the development of new hydrogel chemistry and the maturation of micro- and nano- fabrication. Guided by the spatiotemporal regulation of cell state in developing tissues, we will review the maturation of micropatterning in 2D, pseudo-3D systems, and patterning within 3D hydrogels in the context of translating the information gained from 2D systems to synthetic engineered 3D tissues. PMID:26592366

  7. 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

  8. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  9. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

    PubMed Central

    Riccio, M.; Resca, E.; Maraldi, T.; Pisciotta, A.; Ferrari, A.; Bruzzesi, G.; De Pol, A.

    2010-01-01

    The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects. PMID:21263745

  10. Transcriptional profiles of valvular interstitial cells cultured on tissue culture polystyrene, on 2D hydrogels, or within 3D hydrogels.

    PubMed

    Mabry, Kelly M; Payne, Samuel Z; Anseth, Kristi S

    2015-12-01

    Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis (Chen and Simmons, 2011) [1]. To better understand and quantify how microenvironmental cues influence VIC phenotype, we compared expression profiles of VICs cultured on/in poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. Here, we present both the raw and processed microarray data from these culture conditions. Interpretation of this data can be found in a research article entitled "Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype" (Mabry et al., 2015) [2].

  11. The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance

    PubMed Central

    Breslin, Susan; O'Driscoll, Lorraine

    2016-01-01

    Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs. PMID:27304190

  12. The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.

    PubMed

    Breslin, Susan; O'Driscoll, Lorraine

    2016-07-19

    Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.

  13. Lensfree video microscopy: high throughput monitoring and cell tracking of 2D cell cultures

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Cioni, O.; Momey, F.; Bordy, T.; Hervé, L.; Morel, S.; Navarro, F.; Menneteau, M.; Chalmond, B.; Freida, D.; Sulpice, E.; Gidrol, X.; Dinten, J.-M.

    2015-07-01

    In order to extend the analysis of the datasets produced by lensfree video microscopy we have implemented a cell tracking algorithm to combine and correlate cell motility to the previously devised metrics to quantify e.g. cell adhesion and spreading, cell division, and cell death. In this paper we present the assessment of these new methodology on experiments involving three different cell lines, namely 3T3 fibroblast cells, primary HUVEC cells and macrophage THP1 cells. We demonstrate that the good spatial resolution and the fast frame rate obtained with of our lensfree video microscope allows standard cell tracking algorithm to be computed. The results is the possibility to analyze thousands of cells successfully tracked over tens of hours. The results is the possibility to compare different cell cultures in terms of e.g. cell motility and cell confinement ration. Ultimately we managed to measure the doubling time at single cell level over a large number of N=235 cells tracked over two days.

  14. Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.

    PubMed

    Hara, Jared; Tottori, Jordan; Anders, Megan; Dadhwal, Smritee; Asuri, Prashanth; Mobed-Miremadi, Maryam

    2017-05-01

    Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9 (3(4)) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

  15. Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

    PubMed Central

    Mrozowska, Paulina S.

    2016-01-01

    MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures. PMID:27138252

  16. Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

    SciTech Connect

    Chitcholtan, Kenny; Asselin, Eric; Parent, Sophie; Sykes, Peter H.; Evans, John J.

    2013-01-01

    Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detected by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.

  17. Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.

    PubMed

    Landeira, Bruna Soares; Araújo, Jéssica Alves de Medeiros; Schroeder, Timm; Müller, Ulrich; Costa, Marcos R

    2017-08-09

    During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

  18. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    SciTech Connect

    Ko, Jae Hyung; Kim, Yang Hee; Jeong, Seong Hee; Lee, Song; Park, Si-Nae; Shim, In Kyong; Kim, Song Cheol

    2015-08-07

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

  19. Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures.

    PubMed

    Wang, Tao; Green, Ryan; Nair, Rajesh Ramakrishnan; Howell, Mark; Mohapatra, Subhra; Guldiken, Rasim; Mohapatra, Shyam Sundar

    2015-12-19

    Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose-response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell

  20. Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures

    PubMed Central

    Wang, Tao; Green, Ryan; Nair, Rajesh Ramakrishnan; Howell, Mark; Mohapatra, Subhra; Guldiken, Rasim; Mohapatra, Shyam Sundar

    2015-01-01

    Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell

  1. Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes.

    PubMed

    Tohyama, Shugo; Fujita, Jun; Fujita, Chihana; Yamaguchi, Miho; Kanaami, Sayaka; Ohno, Rei; Sakamoto, Kazuho; Kodama, Masami; Kurokawa, Junko; Kanazawa, Hideaki; Seki, Tomohisa; Kishino, Yoshikazu; Okada, Marina; Nakajima, Kazuaki; Tanosaki, Sho; Someya, Shota; Hirano, Akinori; Kawaguchi, Shinji; Kobayashi, Eiji; Fukuda, Keiichi

    2017-10-04

    Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 10(6) hiPSCs per layer yielded 7.2 × 10(8) hiPSCs in 4-layer CPs and 1.7 × 10(9) hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%-87%. Approximately 6.2-7.0 × 10(8) cells (4-layer) and 1.5-2.8 × 10(9) cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Improving Cardiac Action Potential Measurements: 2D and 3D Cell Culture.

    PubMed

    Daily, Neil J; Yin, Yue; Kemanli, Pinar; Ip, Brian; Wakatsuki, Tetsuro

    2015-11-01

    Progress in the development of assays for measuring cardiac action potential is crucial for the discovery of drugs for treating cardiac disease and assessing cardiotoxicity. Recently, high-throughput methods for assessing action potential using induced pluripotent stem cell (iPSC) derived cardiomyocytes in both two-dimensional monolayer cultures and three-dimensional tissues have been developed. We describe an improved method for assessing cardiac action potential using an ultra-fast cost-effective plate reader with commercially available dyes. Our methods improve dramatically the detection of the fluorescence signal from these dyes and make way for the development of more high-throughput methods for cardiac drug discovery and cardiotoxicity.

  3. Human lung cancer cells grown in an ex vivo 3D lung model produce matrix metalloproteinases not produced in 2D culture.

    PubMed

    Mishra, Dhruva K; Sakamoto, Jason H; Thrall, Michael J; Baird, Brandi N; Blackmon, Shanda H; Ferrari, Mauro; Kurie, Jonathan M; Kim, Min P

    2012-01-01

    We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture.

  4. Regulation of Valvular Interstitial Cell Phenotype and Function by Hyaluronic Acid in 2-D and 3-D Culture Environments

    PubMed Central

    Rodriguez, Karien J.; Piechura, Laura M.; Masters, Kristyn S.

    2010-01-01

    Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2D) and 3D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC-HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC-HA interactions are crucial in maintaining a healthy VIC phenotype. Identification of ECM components that can regulate VIC phenotype and function has significant implications for understanding of native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering. PMID:20884350

  5. Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

    PubMed

    Kamei, Ken-Ichiro; Koyama, Yoshie; Tokunaga, Yumie; Mashimo, Yasumasa; Yoshioka, Momoko; Fockenberg, Christopher; Mosbergen, Rowland; Korn, Othmar; Wells, Christine; Chen, Yong

    2016-11-01

    Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

  6. Backbone Engineered γ-Peptide Amphitropic Gels for Immobilization of Semiconductor Quantum Dots and 2D Cell Culture.

    PubMed

    Misra, Rajkumar; Sharma, Aman; Shiras, Anjali; Gopi, Hosahudya N

    2017-08-08

    We are reporting a spontaneous supramolecular assembly of backbone engineered γ-peptide scaffold and its utility in the immobilization of semiconductor quantum dots and in cell culture. The stimulating feature of this γ-peptide scaffold is that it efficiently gelates both aqueous phosphate buffers and aromatic organic solvents. A comparative and systematic investigation reveals that the greater spontaneous self-aggregation property of γ-peptide over the α- and β-peptide analogues is mainly due to the backbone flexibility, increased hydrophobicity, and π-π stacking of γ-phenylalanine residues. The hydrogels and organogels obtained from the γ-peptide scaffold have been characterized through field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), FT-IR, circular dichroism (CD), wide-angle X-ray diffraction, and rheometric study. Additionally, the peptide hydrogel has displayed a stimuli-responsive and thixotropic signature, which leads to the injectable hydrogels. 2D cell culture studies using normal and cancer cell lines reveal the biocompatibility of γ-peptide hydrogels. Further, the immobilization of semiconductor core-shell quantum dots in the transparent γ-peptide organogels showed ordered arrangement of quantum dots along the peptide fibrillar network with retaining photophysical property. Overall, γ-peptide scaffolds may serve as potential templates for the design of new functional biomaterials.

  7. Development of drug loaded nanoparticles for tumor targeting. Part 1: synthesis, characterization, and biological evaluation in 2D cell cultures

    NASA Astrophysics Data System (ADS)

    El-Dakdouki, Mohammad H.; Puré, Ellen; Huang, Xuefei

    2013-04-01

    Nanoparticles (NPs) are being extensively studied as carriers for drug delivery, but they often have limited penetration inside tumors. We envision that by targeting an endocytic receptor on the cell surface, the uptake of NPs can be significantly enhanced through receptor mediated endocytosis. In addition, if the receptor is recycled to the cell surface, the NP cargo can be transported out of the cells, which is then taken up by neighboring cells thus enhancing solid tumor penetration. To validate our hypothesis, in the first of two articles, we report the synthesis of doxorubicin (DOX)-loaded, hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44, a receptor expressed on the cancer cell surface. HA was conjugated onto amine-functionalized SNPs prepared through an oil-water microemulsion method. The immobilization of the cytotoxic drug DOX was achieved through an acid sensitive hydrazone linkage. The NPs were fully characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, thermogravimetric analysis (TGA), UV-vis absorbance, and nuclear magnetic resonance (NMR). Initial biological evaluation experiments demonstrated that compared to ligand-free SNPs, the uptake of HA-SNPs by the CD44-expressing SKOV-3 ovarian cancer cells was significantly enhanced when evaluated in the 2D monolayer cell culture. Mechanistic studies suggested that cellular uptake of HA-SNPs was mainly through CD44 mediated endocytosis. HA-SNPs with immobilized DOX were endocytosed efficiently by the SKOV-3 cells as well. The enhanced tumor penetration and drug delivery properties of HA-SNPs will be evaluated in 3D tumor models in the subsequent paper.Nanoparticles (NPs) are being extensively studied as carriers for drug delivery, but they often have limited penetration inside tumors. We envision that by targeting an endocytic receptor on the cell surface, the uptake of NPs can be

  8. A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture.

    PubMed

    Zhang, Jue; Schwartz, Michael P; Hou, Zhonggang; Bai, Yongsheng; Ardalani, Hamisha; Swanson, Scott; Steill, John; Ruotti, Victor; Elwell, Angela; Nguyen, Bao Kim; Bolin, Jennifer; Stewart, Ron; Thomson, James A; Murphy, William L

    2017-04-11

    A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for "3D-like" vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    PubMed

    Malenovská, Hana

    2016-02-01

    Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses.

  10. Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype.

    PubMed

    Mabry, Kelly M; Payne, Samuel Z; Anseth, Kristi S

    2016-01-01

    Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis. To better understand and quantify how microenvironmental cues influence VIC phenotype and myofibroblast activation, we compared expression profiles of VICs cultured on poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. In general, VICs cultured in hydrogel matrices had lower levels of activation (<10%), similar to levels seen in healthy valve tissue, while VICs cultured on TCPS were ∼75% activated myofibroblasts. VICs cultured on TCPS also exhibited a higher magnitude of perturbations in gene expression than soft hydrogel cultures when compared to the native phenotype. Using peptide-modified PEG gels, VICs were seeded on (2D), as well as encapsulated in (3D), matrices of the same composition and modulus. Despite similar levels of activation, VICs cultured in 2D had distinct variations in transcriptional profiles compared to those in 3D hydrogels. Genes related to cell structure and motility were particularly affected by the dimensionality of the culture platform, with higher expression levels in 2D than in 3D. These results indicate that dimensionality may play a significant role in dictating cell phenotype (e.g., through differences in polarity, diffusion of soluble signals), and emphasize the importance of using multiple metrics when characterizing cell phenotype.

  11. Regulation of adipose-tissue-derived stromal cell orientation and motility in 2D- and 3D-cultures by direct-current electrical field.

    PubMed

    Yang, Gang; Long, Haiyan; Ren, Xiaomei; Ma, Kunlong; Xiao, Zhenghua; Wang, Ying; Guo, Yingqiang

    2017-02-01

    Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane-protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose-tissue-derived stromal cells (ADSCs) in 2D-culture on plastic culture dishes and in 3D-culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L-lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological-strength EFs in a homemade EF-bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time-lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D-culture aligned vertically to EF vector and kept a good cell survival rate. In 3D-culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D-culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors. © 2017 Japanese Society of Developmental Biologists.

  12. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    NASA Technical Reports Server (NTRS)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  13. Cytotoxic responses of carnosic acid and doxorubicin on breast cancer cells in butterfly-shaped microchips in comparison to 2D and 3D culture.

    PubMed

    Yildiz-Ozturk, Ece; Gulce-Iz, Sultan; Anil, Muge; Yesil-Celiktas, Ozlem

    2017-04-01

    Two dimensional (2D) cell culture systems lack the ability to mimic in vivo conditions resulting in limitations for preclinical cell-based drug and toxicity screening assays and modelling tumor biology. Alternatively, 3D cell culture systems mimic the specificity of native tissue with better physiological integrity. In this regard, microfluidic chips have gained wide applicability for in vitro 3D cancer cell studies. The aim of this research was to develop a 3D biomimetic model comprising culture of breast cancer cells in butterfly-shaped microchip to determine the cytotoxicity of carnosic acid and doxorubicin on both estrogen dependent (MCF-7) and independent (MDA-MB231) breast cancer cells along with healthy mammary epithelial cells (MCF-10A) in 2D, 3D Matrigel™ and butterfly-shaped microchip environment. According to the developed mimetic model, carnosic acid exhibited a higher cytotoxicity towards MDA-MB 231, while doxorubicin was more effective against MCF-7. Although the cell viabilities were higher in comparison to 2D and 3D cell culture systems, the responses of the investigated molecules were different in the microchips based on the molecular weight and structural complexity indicating the importance of biomimicry in a physiologically relevant matrix.

  14. Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems.

    PubMed

    Brajša, Karmen; Vujasinović, Ines; Jelić, Dubravko; Trzun, Marija; Zlatar, Ivo; Karminski-Zamola, Grace; Hranjec, Marijana

    2016-12-01

    Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure-activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.

  15. Comparison of several radiation effects in human MCF10A mammary epithelial cells cultured as 2D monolayers or 3D acinar stuctures in matrigel.

    PubMed

    Lin, Yu-Fen; Nagasawa, Hatsumi; Peng, Yuanlin; Chuang, Eric Y; Bedford, Joel S

    2009-06-01

    It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very differently than the same cells in a 2D monolayer culture. While many studies have shown that, in some cases, cell-cell contact in spheroids of transformed or tumor cell lines can alter radiation responses relative to those for the same cells in monolayer cultures, a question remains regarding the possible effect of the above-mentioned disruption of signaling networks that operate more specifically for cells in normal tissues or in a 3D tissue-like context. To test the generality of this notion, we used human MCF-10A cells, an immortalized mammary epithelial cell line that produces acinar structures in culture with many properties of human mammary ducts. We compared the dose responses for these cells in the 2D monolayer and in 3D ductal or acinar structures. The responses examined were reproductive cell death, induction of chromosomal aberrations, and the levels of gamma-H2AX foci in cells after single acute gamma-ray doses and immediately after 20 h of irradiation at a dose rate of 0.0017 Gy/min. We found no significant differences in the dose responses of these cells in 2D or 3D growth conditions. While this does not mean that such differences cannot occur in other situations, it does mean that they do not generally or necessarily occur.

  16. Differential effects of MAPK pathway inhibitors on migration and invasiveness of BRAF(V600E) mutant thyroid cancer cells in 2D and 3D culture.

    PubMed

    Ingeson-Carlsson, Camilla; Martinez-Monleon, Angela; Nilsson, Mikael

    2015-11-01

    Tumor microenvironment influences targeted drug therapy. In this study we compared drug responses to RAF and MEK inhibitors on tumor cell migration in 2D and 3D culture of BRAF(V600E) mutant cell lines derived from human papillary (BCPAP) and anaplastic (SW1736) thyroid carcinomas. Scratch wounding was compared to a double-layered collagen gel model developed for analysis of directed tumor cell invasion during prolonged culture. In BCPAP both PLX4720 and U0126 inhibited growth and migration in 2D and decreased tumor cell survival in 3D. In SW1736 drugs had no effect on migration in 2D but decreased invasion in 3D, however this related to reduced growth. Dual inhibition of BRAF(V600E) and MEK reduced but did not prevent SW1736 invasion although rebound phosphorylation of ERK in response to PLX4720 was blocked by U0126. These findings indicate that anti-tumor drug effects in vitro differ depending on culture conditions (2D vs. 3D) and that the invasive features of anaplastic thyroid cancer depend on non-MEK mechanism(s).

  17. The mechanical coupling of adult marrow stromal stem cells during cardiac regeneration assessed in a 2-D co-culture model.

    PubMed

    Valarmathi, Mani T; Fuseler, John W; Goodwin, Richard L; Davis, Jeffrey M; Potts, Jay D

    2011-04-01

    Postnatal cardiomyocytes undergo terminal differentiation and a restricted number of human cardiomyocytes retain the ability to divide and regenerate in response to ischemic injury. However, whether these neo-cardiomyocytes are derived from endogenous population of resident cardiac stem cells or from the exogenous double assurance population of resident bone marrow-derived stem cells that populate the damaged myocardium is unresolved and under intense investigation. The vital challenge is to ameliorate and/or regenerate the damaged myocardium. This can be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or allogeneic cells such as fetal or embryonic cardiomyocyte progenitors or bone marrow-derived stromal stem cells. The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the native myocardium and must exhibit functional synchronization. Adult bone marrow stromal cells (BMSCs) have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in cardiac repair and regeneration, but this concept requires further validation. In this report, we have provided compelling evidence that functioning cardiac tissue can be generated by the interaction of multipotent BMSCs with embryonic cardiac myocytes (ECMs) in two-dimensional (2-D) co-cultures. The differentiating BMSCs were induced to undergo cardiomyogenic differentiation pathway and were able to express unequivocal electromechanical coupling and functional synchronization with ECMs. Our 2-D co-culture system provides a useful in vitro model to elucidate various molecular mechanisms underpinning the integration and orderly maturation and differentiation of BMSCs into neo-cardiomyocytes during myocardial repair and regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Phototoxic action of a zinc(II) phthalocyanine encapsulated into poloxamine polymeric micelles in 2D and 3D colon carcinoma cell cultures.

    PubMed

    Chiarante, Nicolás; García Vior, María C; Awruch, Josefina; Marino, Julieta; Roguin, Leonor P

    2017-05-01

    Photodynamic therapy is emerging as a hopeful method for the treatment of oncological diseases. In the search of novel therapeutic strategies for colorectal cancer, in this work we reported the photocytotoxic activity of a lipophilic zinc(II) phthalocyanine on a murine colon adenocarcinoma cell line (CT26 cells). The 2,9(10),16(17),23(24) tetrakis[(2-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II), named Pc9, was encapsulated into Tetronic® 1107 polymeric poloxamine micelles (T1107) and assayed in 2D and 3D cell cultures. We showed that the formulation Pc9-T1107 was efficient to reduce cell viability after photodynamic treatment both in 2D cultures (IC50 10±2nM) as well as in CT26 spheroids (IC50 370±11nM). Cellular uptake of Pc9-T1107 was a time- and concentration-dependent process, being the phthalocyanine formulation mainly incorporated into lysosomal vesicles and endoplasmic reticulum cisterns, but not in mitochondria. Pc9-T1107 also induced the formation of reactive oxygen species immediately after cell irradiation. We also found that the phototoxic action of Pc9-T1107 was partially reversed in the presence of antioxidants, such as TROLOX and N-acetyl-cysteine. In addition, we showed that Pc9-T1107 treatment triggered an apoptotic cell death, as suggested by the detection of pyknotic nuclei, the reduction in the expression levels of procaspase-3 and the increase in caspase-3 enzymatic activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Spatio-temporal morphology changes in and quenching effects on the 2D spreading dynamics of cell colonies in both plain and methylcellulose-containing culture media.

    PubMed

    Muzzio, N E; Pasquale, M A; Huergo, M A C; Bolzán, A E; González, P H; Arvia, A J

    2016-06-01

    To deal with complex systems, microscopic and global approaches become of particular interest. Our previous results from the dynamics of large cell colonies indicated that their 2D front roughness dynamics is compatible with the standard Kardar-Parisi-Zhang (KPZ) or the quenched KPZ equations either in plain or methylcellulose (MC)-containing gel culture media, respectively. In both cases, the influence of a non-uniform distribution of the colony constituents was significant. These results encouraged us to investigate the overall dynamics of those systems considering the morphology and size, the duplication rate, and the motility of single cells. For this purpose, colonies with different cell populations (N) exhibiting quasi-circular and quasi-linear growth fronts in plain and MC-containing culture media are investigated. For small N, the average radial front velocity and its change with time depend on MC concentration. MC in the medium interferes with cell mitosis, contributes to the local enlargement of cells, and increases the distribution of spatio-temporal cell density heterogeneities. Colony spreading in MC-containing media proceeds under two main quenching effects, I and II; the former mainly depending on the culture medium composition and structure and the latter caused by the distribution of enlarged local cell domains. For large N, colony spreading occurs at constant velocity. The characteristics of cell motility, assessed by measuring their trajectories and the corresponding velocity field, reflect the effect of enlarged, slow-moving cells and the structure of the medium. Local average cell size distribution and individual cell motility data from plain and MC-containing media are qualitatively consistent with the predictions of both the extended cellular Potts models and the observed transition of the front roughness dynamics from a standard KPZ to a quenched KPZ. In this case, quenching effects I and II cooperate and give rise to the quenched

  20. Evaluation of human cord blood CD34+ hematopoietic stem cell differentiation to megakaryocyte on aminated PES nanofiber scaffold compare to 2-D culture system.

    PubMed

    Sabaghi, Fatemeh; Shamsasenjan, Karim; Movasaghpour, Ali Akbari; Amirizadeh, Naser; Nikougoftar, Mahin; Bagheri, Nadia

    2016-06-01

    Recently, umbilical cord blood (UCB) has been recognized as a suitable potential source of hematopoietic stem/progenitor cells (HSPCs) for transplantation. Lengthy thrombocytopenia after UCB transplantation is a major problem because of insufficient megakaryocyte (Mk) progenitors, which results in delayed platelet recovery. Frequent allogenic platelet transfusion leads to resistance to platelet units and higher risk of transmission of pathogenic agent. Ex vivo expansion of HSPCs and their differentiation to Mk progenitors on aminated PES nanofiber could lead to faster platelet recovery after UCB transplantation. CD34 cells were positively enriched using the MidiMACS system. CD34(+) cells were seeded onto conventional culture and aminated PES scaffold. The proliferation of CD34(+) cells, and also their differentiation into Mk progenitors, were evaluated. We used the flow cytometric method for analyzing CD41 and CD61 markers and real-time PCR for the expression level of transcription factors, as NF-E2 and GATA-1. This study indicated increased CD34(+) cell population in aminated PES compared to the conventional system. After differentiation, the amount of CD41/CD61-expressing cells and the quantity of NF-E2 expression level increased in the aminated PES versus the 2-D system. The quantity of GATA-1 expression level was reduced on CD41/CD61(+) cells compared to CD34(+) cells, with no difference between the aminated PES and the conventional system. Aminated PES nanofiber could have more effect on the proliferation of CD34(+) cells and Mk differentiation than the conventional culture. Injection of the expanded cells and differentiated Mk progenitors, along with the transplantation of UCB stem cells might accelerate recovery of platelets and decrease the period of thrombocytopenia after transplantation.

  1. Expression of transcription factors after short-term exposure of Arabidopsis thaliana cell cultures to hypergravity and simulated microgravity (2-D/3-D clinorotation, magnetic levitation)

    NASA Astrophysics Data System (ADS)

    Babbick, M.; Dijkstra, C.; Larkin, O. J.; Anthony, P.; Davey, M. R.; Power, J. B.; Lowe, K. C.; Cogoli-Greuter, M.; Hampp, R.

    Gravity is an important environmental factor that controls plant growth and development. Studies have shown that the perception of gravity is not only a property of specialized cells, but can also be performed by undifferentiated cultured cells. In this investigation, callus of Arabidopsis thaliana cv. Columbia was used to investigate the initial steps of gravity-related signalling cascades, through altered expression of transcription factors (TFs). TFs are families of small proteins that regulate gene expression by binding to specific promoter sequences. Based on microarray studies, members of the gene families WRKY, MADS-box, MYB, and AP2/EREBP were selected for investigation, as well as members of signalling chains, namely IAA 19 and phosphoinositol-4-kinase. Using qRT-PCR, transcripts were quantified within a period of 30 min in response to hypergravity (8 g), clinorotation [2-D clinostat and 3-D random positioning machine (RPM)] and magnetic levitation (ML). The data indicated that (1) changes in gravity induced stress-related signalling, and (2) exposure in the RPM induced changes in gene expression which resemble those of magnetic levitation. Two dimensional clinorotation resulted in responses similar to those caused by hypergravity. It is suggested that RPM and ML are preferable to simulate microgravity than clinorotation.

  2. Binding of galectin-1 to breast cancer cells MCF7 induces apoptosis and inhibition of proliferation in vitro in a 2D- and 3D- cell culture model.

    PubMed

    Geiger, Pamina; Mayer, Barbara; Wiest, Irmi; Schulze, Sandra; Jeschke, Udo; Weissenbacher, Tobias

    2016-11-08

    Galectin-1 (gal-1) belongs to the family of β-galactoside-binding proteins which primarily recognizes the Galβ1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. The lectin recognizes correspondent glycoepitopes on human breast cancer cells. Galectin-1 is expressed both in normal and malignant tissues. Lymphatic organs naturally possessing high rates of apoptotic cells, express high levels of Galectin-1. Furthermore galectin-1 can initiate T cell apoptosis. Binding of galectin-1 to trophoblast tumor cells presenting the oncofetal Thomsen-Friedenreich (TF) carbohydrate antigen inhibits tumor cell proliferation. In this study we examined the impact galectin-1 has in vitro on cell proliferation, apoptotic potential and metabolic activity of MCF-7 and T-47D breast cancer cells in dependence to their expression of the Thomsen-Friedenreich (TF) tumor antigen. For proliferation and apoptosis assays cells were grown in presence of 10, 30 and 60 μg gal-1/ml medium. Cell proliferation was determined by a BrdU uptake ELISA. Detection of apoptotic cells was done by M30 cyto death staining, in situ nick translation and by a nucleosome ELISA method. Furthermore we studied the impact galectin-1 has on the metabolic activity of MCF-7 and T-47D cells in a homotypic three-dimensional spheroid cell culture model mimicking a micro tumour environment. Gal-1 inhibited proliferation of MCF-7 cells (strong expression of the TF epitope) but did not significantly change proliferation of T-47D cells (weak expression of the TF epitope). The incubation of MCF-7 cells with gal-1 raised number of apoptotic cells significantly. Treating the spheroids with 30 μg/ml galectin-1 in addition to standard chemotherapeutic regimes (FEC, TAC) resulted in further suppression of the metabolic activity in MCF-7 cells whereas T-47D cells were not affected. Our results demonstrate that galectin-1 can inhibit proliferation und metabolic cell activity and induce

  3. Traditional Chinese medicine herbal mixture LQ arrests FUCCI-expressing HeLa cells in G₀/G₁ phase in 2D plastic, 2.5D Matrigel, and 3D Gelfoam culture visualized with FUCCI imaging.

    PubMed

    Zhang, Lei; Wu, Chengyu; Bouvet, Michael; Yano, Shuya; Hoffman, Robert M

    2015-03-10

    We used the fluorescence ubiquitination-based cell cycle indicator (FUCCI) to monitor cell cycle arrest after treatment of FUCCI-expressing HeLa cells (FUCCI-HeLa) with a traditional Chinese medicine (TCM) herbal mixture LQ, previously shown to have anti-tumor and anti-metastatic activity in mouse models. Paclitaxel was used as the positive control. In 2D monolayer culture, the untreated control had approximately 45% of the cells in S/G₂/M phase. In contrast, the LQ-treated cells (9 mg/ml) were mostly in the G₀/G₁ (>90%) after 72 hours. After treatment with paclitaxel (0.01 μm), for 72 hours, 95% of the cells were in S/G₂/M. In 2.5D Matrigel culture, the colonies in the untreated control group had 40% of the cells in S/G₂/M. LQ arrested the cells in G₀/G₁ after 72 hours. Paclitaxel arrested almost all the cells in S/G₂/M after 72 hours. In 3D Gelfoam culture, the untreated control culture had approximately 45% of cells in G₂/M. In contrast, the LQ-treated cells were mostly in G₀/G₁ phase (>80%) after 72 hours treatment. Paclitaxel resulted in 90% of the cells arrested in S/G₂/M after 72 hours. The present report suggests the non-toxic LQ has potential to maintain cancers in a quiescent state for long periods of time.

  4. Systemic administration of cell-free exosomes generated by human bone marrow derived mesenchymal stem cells cultured under 2D and 3D conditions improves functional recovery in rats after traumatic brain injury.

    PubMed

    Zhang, Yanlu; Chopp, Michael; Zhang, Zheng Gang; Katakowski, Mark; Xin, Hongqi; Qu, Changsheng; Ali, Meser; Mahmood, Asim; Xiong, Ye

    2016-08-15

    Multipotent human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after experimental traumatic brain injury (TBI). The present study was designed to investigate whether systemic administration of cell-free exosomes generated from hMSCs cultured in 2-dimensional (2D) conventional conditions or in 3-dimensional (3D) collagen scaffolds promote functional recovery and neurovascular remodeling in rats after TBI. Wistar rats were subjected to TBI induced by controlled cortical impact; 24 h later tail vein injection of exosomes derived from hMSCs cultured under 2D or 3D conditions or an equal number of liposomes as a treatment control were performed. The modified Morris water maze, neurological severity score and footfault tests were employed to evaluate cognitive and sensorimotor functional recovery. Animals were sacrificed at 35 days after TBI. Histological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. Compared with liposome-treated control, exosome-treatments did not reduce lesion size but significantly improved spatial learning at 33-35 days measured by the Morris water maze test, and sensorimotor functional recovery, i.e., reduced neurological deficits and footfault frequency, observed at 14-35 days post injury (p < 0.05). Exosome treatments significantly increased the number of newborn endothelial cells in the lesion boundary zone and dentate gyrus, and significantly increased the number of newborn mature neurons in the dentate gyrus as well as reduced neuroinflammation. Exosomes derived from hMSCs cultured in 3D scaffolds provided better outcome in spatial learning than exosomes from hMSCs cultured in the 2D condition. In conclusion, hMSC-generated exosomes significantly improve functional recovery in rats after TBI, at least in part, by promoting endogenous angiogenesis and neurogenesis and reducing

  5. Alternatively spliced MEFV transcript lacking exon 2 and its protein isoform pyrin-2d implies an epigenetic regulation of the gene in inflammatory cell culture models

    PubMed Central

    Erdem, Gokce Celikyapi; Erdemir, Sule; Abaci, Irem; Aydin, Asli K. Kirectepe; Everest, Elif; Turanli, Eda Tahir

    2017-01-01

    Abstract The function of gene body DNA methylation in alternative splicing, and its relation to disease pathogenesis is not fully elucidated. The gene for familial Mediterranean fever (MEFV) encodes the pyrin protein and contains a 998 bp CpG island, covering the second exon, which is differentially methylated in FMF patients compared to healthy controls. Our further observation of increased exon 2-spliced MEFV transcript in leukocytes of FMF patients provoked us to test the role of exon methylation in alternative splicing using inflammatory cell culture models. First, in vitro exon methylation triggered an increased level of exon 2 exclusion using a splicing cassette in a promyelocytic leukemia cell line (HL-60). HL-60 cells subjected to methylating and demethylating agents, as well as cells differentiated to neutrophil-like cells, exhibited different levels of spliced/unspliced transcripts. We observed increased levels of spliced transcripts in neutrophil-like (p = 0.0005), activated (p = 0.0034) and methylated cells (p < 0.0001), whereas decreased levels in demethylated cells (p = 0.0126) compared to control untreated HL-60 cells. We also showed that the protein isoform of pyrin lacking the exon 2 has an adverse subcellular localization in neutrophil-like cells. Therefore, it remains in the cytoplasm rather than the nucleus. This may point to an epigenetic involvement in an important inflammatory gene. PMID:28863210

  6. Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture.

    PubMed

    Yu, Miao; Huang, Shaohui; Yu, Kevin Jun; Clyne, Alisa Morss

    2012-01-01

    Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

  7. Dextran and Polymer Polyethylene Glycol (PEG) Coating Reduce Both 5 and 30 nm Iron Oxide Nanoparticle Cytotoxicity in 2D and 3D Cell Culture

    PubMed Central

    Yu, Miao; Huang, Shaohui; Yu, Kevin Jun; Clyne, Alisa Morss

    2012-01-01

    Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles. PMID:22754315

  8. Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

    PubMed Central

    2012-01-01

    Background Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity. Results We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells. Conclusions Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells. PMID:22316211

  9. Antiproliferative activity of amino substituted benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles explored by 2D and 3D cell culture system.

    PubMed

    Perin, Nataša; Bobanović, Kristina; Zlatar, Ivo; Jelić, Dubravko; Kelava, Vanja; Koštrun, Sanja; Marković, Vesna Gabelica; Brajša, Karmen; Hranjec, Marijana

    2017-01-05

    Benzimidazo[1,2-a]quinolines and benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles with amino chains on the different positions have been evaluated by 2D and 3D assays on the human breast cancer cells. Pentacyclic derivatives were synthesized by microwave assisted amination to study the influence of the thiophene substructure on antitumor activity in comparison to tetracyclic analogues. The results obtained from 2D assay reveals that the antitumor activity is strongly dependent on the nature and position of amino chains. Tetracyclic derivatives displayed selective activity on SK-BR-3 with the 2-amino substituted derivatives as the most active ones while pentacyclic derivatives 6-16 and 21-25 showed more pronounced activity on T-47D. The evaluation of antitumor activity in the 3D assay pointed out that some of the tetracyclic and pentacyclic amino substituted derivatives showed selective activity on the MDA-MB-231 cell line. Influence of physico-chemical properties of the compounds on antiproliferative activity have been investigated by multivariate statistical methods. As a measure of lipophilicity, experimental Chrom LogD values have been determined and number of structural parameters have been calculated for investigated compounds. Main factors contributing to the antiproliferative effect for both 2D and 3D cell cultures are found to be basicity, lipophilicity, molecular weight and number of H-bond donors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. 2D and 3D Self-Assembling Nanofiber Hydrogels for Cardiomyocyte Culture

    PubMed Central

    Ikonen, Liisa; Kerkelä, Erja; Metselaar, Gerald; Stuart, Marc C. A.; de Jong, Menno R.; Aalto-Setälä, Katriina

    2013-01-01

    Collagen is a widely used biomaterial in cardiac tissue engineering studies. However, as a natural material, it suffers from variability between batches that can complicate the standardization of culture conditions. In contrast, synthetic materials are modifiable, have well-defined structures and more homogeneous batches can be produced. In this study, several collagen-like synthetic self-assembling nanofiber hydrogels were examined for their suitability for cardiomyocyte culture in 2D and 3D. Six different nanofiber coatings were used in the 2D format with neonatal rat cardiomyocytes (NRCs) and human embryonic stem-cell-derived cardiomyocytes (hESC-CMs). The viability, growth, and functionality of the 2D-cultured cardiomyocytes were evaluated. The best-performing nanofiber coatings were selected for 3D experiments. Hydrophilic pH-sensitive nanofiber hydrogel coassembled with hyaluronic acid performed best with both NRCs and hESC-CMs. Hydrophilic non-pH-sensitive nanofiber hydrogels supported the growth of NRCs; however, their ability to promote attachment and growth of hESC-CMs was limited. NRCs also grew on hydrophobic nanofiber hydrogels; however, the cell-supporting capacity of these hydrogels was inferior to that of the hydrophilic hydrogel materials. This is the first study demonstrating that hydrophilic self-assembling nanofiber hydrogels support the culture of both NRCs and hESC-CMs, which suggests that these biomaterials hold promise for cardiac tissue engineering. PMID:23573513

  11. NKG2D ligands mediate immunosurveillance of senescent cells

    PubMed Central

    Moshayev, Zhana; Vadai, Ezra; Wensveen, Felix; Ben-Dor, Shifra; Golani, Ofra; Polic, Bojan; Krizhanovsky, Valery

    2016-01-01

    Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis. Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis. PMID:26878797

  12. Polychromatic light-induced osteogenic activity in 2D and 3D cultures.

    PubMed

    Ülker, Nazife; Çakmak, Anıl S; Kiremitçi, Arlin S; Gümüşderelioğlu, Menemşe

    2016-11-01

    Photobiomodulation (PBM) has been applied to manipulate cellular responses by using monochromatic light in different wavelengths from ultraviolet (UV) to infrared (IR) region. Until now, an effective wavelength has not been revealed to induce proliferation and/or differentiation of cells. Therefore, in the presented study, we decided to use a specially designed plasma arc light source providing wavelengths between 590 and 1500 nm in order to investigate its biomodulatory effects on chitosan scaffold-supported three-dimensional (3D) cell cultures. For comparison, two-dimensional (2D) cell cultures were also carried out in tissue-culture polystyrene dishes (TCPS). The results showed that light-induced temperature rise did not affect cells when the distance between the light source and the cells was 10 cm and the frequency of administration was daily. Moreover, light was applied for 5 and 10 min to the cells in TCPS and in chitosan scaffold groups, respectively. Cell culture studies under static conditions indicated that polychromatic light significantly stimulated bone nodule formation via the prolonged cell survival and stimulated differentiation of MC3T3-E1 preosteoblastic cells in both TCPS and chitosan scaffold groups. In conclusion, specially designed plasma arc light source used in this study induces formation of bone tissue and so, this light source is proposed as an appropriate system for in vitro bone tissue engineering applications. Statistical analyses were performed with one-way ANOVA by using GraphPad Instat software and standard deviations were calculated by using data of three parallel samples for each group.

  13. ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing.

    PubMed

    Zocchi, Maria Raffaella; Camodeca, Caterina; Nuti, Elisa; Rossello, Armando; Venè, Roberta; Tosetti, Francesca; Dapino, Irene; Costa, Delfina; Musso, Alessandra; Poggi, Alessandro

    2016-05-01

    Hodgkin lymphoma (HL) resistant to conventional therapies is increasing, making of interest the search for new schemes of treatment. Members of the "A Disintegrin And Metalloproteases" (ADAMs) family, mainly ADAM10 or ADAM17, have been proposed as therapeutic targets in solid tumors and some ADAMs inhibitors have been shown to exert antitumor effects. We have previously described an overexpression of ADAM10 in HL, together with increased release of NKG2D ligands (NKG2D-L) and reduced activation of effector T lymphocytes with anti-lymphoma capacity. Aim of the present work was to verify whether inhibition of ADAM10 in HL cells could restore the triggering of NKG2D-dependent anti-lymphoma T cell response. As no selective ADAM10 blockers have been reported so far, we synthesized the two hydroxamate compounds LT4 and MN8 with selectivity for ADAM10 over metalloproteases (MMPs), LT4 showing higher specificity for ADAM10 over ADAM17. We show that (i) HL lymph nodes (LN) and cultured HL cells express high levels of the mature active membrane form of ADAM10; (ii) ADAM10 is the major sheddase for the NKG2D-L in HL cells; (iii) the new LT4 and MN8 compounds strongly reduce the shedding of NKG2D-L by HL cell lines and enhance the binding of NKG2D receptor; (iv) of note, these new ADAM10 inhibitors increase the sensitivity of HL cell lines to NKG2D-dependent cell killing exerted by natural killer and γδ T cells. Overall, the biologic activity of LT4 and MN8 appears to be more potent than that of the commercial inhibitor GI254023X.

  14. ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

    PubMed Central

    Zocchi, Maria Raffaella; Camodeca, Caterina; Nuti, Elisa; Rossello, Armando; Venè, Roberta; Tosetti, Francesca; Dapino, Irene; Costa, Delfina; Musso, Alessandra; Poggi, Alessandro

    2016-01-01

    ABSTRACT Hodgkin lymphoma (HL) resistant to conventional therapies is increasing, making of interest the search for new schemes of treatment. Members of the “A Disintegrin And Metalloproteases” (ADAMs) family, mainly ADAM10 or ADAM17, have been proposed as therapeutic targets in solid tumors and some ADAMs inhibitors have been shown to exert antitumor effects. We have previously described an overexpression of ADAM10 in HL, together with increased release of NKG2D ligands (NKG2D-L) and reduced activation of effector T lymphocytes with anti-lymphoma capacity. Aim of the present work was to verify whether inhibition of ADAM10 in HL cells could restore the triggering of NKG2D-dependent anti-lymphoma T cell response. As no selective ADAM10 blockers have been reported so far, we synthesized the two hydroxamate compounds LT4 and MN8 with selectivity for ADAM10 over metalloproteases (MMPs), LT4 showing higher specificity for ADAM10 over ADAM17. We show that (i) HL lymph nodes (LN) and cultured HL cells express high levels of the mature active membrane form of ADAM10; (ii) ADAM10 is the major sheddase for the NKG2D-L in HL cells; (iii) the new LT4 and MN8 compounds strongly reduce the shedding of NKG2D-L by HL cell lines and enhance the binding of NKG2D receptor; (iv) of note, these new ADAM10 inhibitors increase the sensitivity of HL cell lines to NKG2D-dependent cell killing exerted by natural killer and γδ T cells. Overall, the biologic activity of LT4 and MN8 appears to be more potent than that of the commercial inhibitor GI254023X. PMID:27467923

  15. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

    PubMed Central

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

    2016-01-01

    This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

  16. The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments

    PubMed Central

    Kanlaya, Rattiyaporn; Borkowski, Kamil; Schwämmle, Veit; Dai, Jie; Joensen, Kira Eyd; Wojdyla, Katarzyna; Carvalho, Vasco Botelho; Fey, Stephen J.

    2014-01-01

    Introduction Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Results Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. Summary We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance. PMID:25222612

  17. Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells.

    PubMed

    Marchionatti, Ana M; Picotto, Gabriela; Narvaez, Carmen J; Welsh, Joellen; Tolosa de Talamoni, Nori G

    2009-02-01

    Calcitriol or 1,25(OH)(2)D(3) is a negative growth regulator of MCF-7 breast cancer cells. The growth arrest is due to apoptosis activation, which involves mitochondrial disruption. This effect is blunted in vitamin D resistant cells (MCF-7(DRes) cells). Menadione (MEN), a glutathione (GSH)-depleting compound, may potentiate antitumoral effects of anticancer drugs. The aim of this study was to investigate whether MEN enhances cellular responsiveness of MCF-7 cells to 1,25(OH)(2)D(3). Cells were cultured and treated with different concentrations of 1,25(OH)(2)D(3)+/-MEN or vehicle for 96 h. GSH levels and the activity of antioxidant enzymes were determined by spectrophotometry and ROS production by flow cytometry. Both drugs decreased growth and enhanced ROS in MCF-7 cells, obtaining the maximal effects when 1,25(OH)(2)D(3) was combined with MEN (P<0.01 vs. Control and vs. each compound alone). MCF-7(DRes) cells were not responsive to 1,25(OH)(2)D(3), but the cell proliferation was slightly inhibited by the combined treatment. Calcitriol and MEN separately enhanced antioxidant enzyme activities, but when they were used in combination, the effect was more pronounced (P<0.05 vs. Control and vs. each compound alone). MEN, calcitriol and the combined treatment decreased GSH levels (P<0.05 vs. Control). The data indicate that MEN potentiates the effect of 1,25(OH)(2)D(3) on growth arrest in MCF-7 cells by oxidative stress and increases the activities of antioxidant enzymes, probably as a compensatory mechanism.

  18. Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices.

    PubMed

    Casey, A; Gargotti, M; Bonnier, F; Byrne, H J

    2016-06-01

    The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in "reduced" toxicity or cellular "resistance" to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug

  19. 2-D Model for Normal and Sickle Cell Blood Microcirculation

    NASA Astrophysics Data System (ADS)

    Tekleab, Yonatan; Harris, Wesley

    2011-11-01

    Sickle cell disease (SCD) is a genetic disorder that alters the red blood cell (RBC) structure and function such that hemoglobin (Hb) cannot effectively bind and release oxygen. Previous computational models have been designed to study the microcirculation for insight into blood disorders such as SCD. Our novel 2-D computational model represents a fast, time efficient method developed to analyze flow dynamics, O2 diffusion, and cell deformation in the microcirculation. The model uses a finite difference, Crank-Nicholson scheme to compute the flow and O2 concentration, and the level set computational method to advect the RBC membrane on a staggered grid. Several sets of initial and boundary conditions were tested. Simulation data indicate a few parameters to be significant in the perturbation of the blood flow and O2 concentration profiles. Specifically, the Hill coefficient, arterial O2 partial pressure, O2 partial pressure at 50% Hb saturation, and cell membrane stiffness are significant factors. Results were found to be consistent with those of Le Floch [2010] and Secomb [2006].

  20. Tumour-experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

    PubMed Central

    Domaica, Carolina I; Fuertes, Mercedes B; Rossi, Lucas E; Girart, María V; Ávila, Damián E; Rabinovich, Gabriel A; Zwirner, Norberto W

    2009-01-01

    Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-γ secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co-culture with some melanomas, activated CD4+ T cells promoted degranulation, and NKG2D- and NKp46-dependent IFN-γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4+, CD8+ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. PMID:19498463

  1. Memory T Cells Expressing an NKG2D-CAR Efficiently Target Osteosarcoma Cells.

    PubMed

    Fernández, Lucía; Metais, Jean-Yves; Escudero, Adela; Vela, María; Valentín, Jaime; Vallcorba, Isabel; Leivas, Alejandra; Torres, Juan; Valeri, Antonio; Patiño-García, Ana; Martínez, Joaquín; Leung, Wing; Pérez-Martínez, Antonio

    2017-10-01

    Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8(+) T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA(-) memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR).Experimental Design: CD45RA(-) cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR(+) CD45RA(-) T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rg(null) mice.Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA(-) cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR(+) memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR(+) memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective.Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824-35. ©2017 AACR. ©2017 American Association for Cancer Research.

  2. CD4+ NKG2D+ T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1ε transgenic mice

    PubMed Central

    Lin, Zhijie; Wang, Changrong; Xia, Haizui; Liu, Weiguang; Xiao, Weiming; Qian, Li; Jia, Xiaoqin; Ding, Yanbing; Ji, Mingchun; Gong, Weijuan

    2014-01-01

    The binding of NKG2D to its ligands strengthens the cross-talk between natural killer (NK) cells and dendritic cells, particularly at early stages, before the initiation of the adaptive immune response. We found that retinoic acid early transcript-1ε (RAE-1ε), one of the ligands of NKG2D, was persistently expressed on antigen-presenting cells in a transgenic mouse model (pCD86-RAE-1ε). By contrast, NKG2D expression on NK cells, NKG2D-dependent cytotoxicity and tumour rejection, and dextran sodium sulphate-induced colitis were all down-regulated in this mouse model. The down-regulation of NKG2D on NK cells was reversed by stimulation with poly (I:C). The ectopic expression of RAE-1ε on dendritic cells maintained NKG2D expression levels and stimulated the activity of NK cells ex vivo, but the higher frequency of CD4+ NKG2D+ T cells in transgenic mice led to the down-regulation of NKG2D on NK cells in vivo. Hence, high levels of RAE-1ε expression on antigen-presenting cells would be expected to induce the down-regulation of NK cell activation by a regulatory T-cell subset. PMID:24708417

  3. CD4(+) NKG2D(+) T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1ε transgenic mice.

    PubMed

    Lin, Zhijie; Wang, Changrong; Xia, Haizui; Liu, Weiguang; Xiao, Weiming; Qian, Li; Jia, Xiaoqin; Ding, Yanbing; Ji, Mingchun; Gong, Weijuan

    2014-03-01

    The binding of NKG2D to its ligands strengthens the cross-talk between natural killer (NK) cells and dendritic cells, particularly at early stages, before the initiation of the adaptive immune response. We found that retinoic acid early transcript-1ε (RAE-1ε), one of the ligands of NKG2D, was persistently expressed on antigen-presenting cells in a transgenic mouse model (pCD86-RAE-1ε). By contrast, NKG2D expression on NK cells, NKG2D-dependent cytotoxicity and tumour rejection, and dextran sodium sulphate-induced colitis were all down-regulated in this mouse model. The down-regulation of NKG2D on NK cells was reversed by stimulation with poly (I:C). The ectopic expression of RAE-1ε on dendritic cells maintained NKG2D expression levels and stimulated the activity of NK cells ex vivo, but the higher frequency of CD4(+) NKG2D(+) T cells in transgenic mice led to the down-regulation of NKG2D on NK cells in vivo. Hence, high levels of RAE-1ε expression on antigen-presenting cells would be expected to induce the down-regulation of NK cell activation by a regulatory T-cell subset.

  4. Behaviour of gravisensitive cells on 2D and 3D clinostats

    NASA Astrophysics Data System (ADS)

    Strauch, Sebastian M.; Hemmersbach, Ruth; Seibt, Dieter; Schuber, Marianne; Hader, Donat-P.

    2005-08-01

    2D and 3D clinostats are widely applied to study the influence of simulated microgravity on different kinds of organisms and cell cultures [1]. To critically evaluate the results achieved (functional weightlessness, omnilateral gravistimulation or other side effects such as strong mechanical disturbances) a comparison between the applied simulation methods and real microgravity is necessary. In a first approach, the swimming behavior of Euglena gracilis, a "professional gravi-sensing" unicellular freshwater flagellate, was observed under 2D and 3D clinostat conditions as well as under real microgravity during a TEXUS sounding rocket flight.According to current theory Euglena perceives the gravity vector by stimulation of mechanosensitive channels: the cell mass, which is denser than the surrounding medium, exerts pressure onto the lower membrane and the resulting gated calcium influx modulates the beating pattern of the flagella [4].A changed influence of gravity of the cells can be directly visualized by changes in their orientation with respect to gravity (gravitaxis).

  5. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    NASA Astrophysics Data System (ADS)

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-03-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.

  6. Activating receptor NKG2D targets RAE-1-expressing allogeneic neural precursor cells in a viral model of multiple sclerosis.

    PubMed

    Weinger, Jason G; Plaisted, Warren C; Maciejewski, Sonia M; Lanier, Lewis L; Walsh, Craig M; Lane, Thomas E

    2014-10-01

    Transplantation of major histocompatibility complex-mismatched mouse neural precursor cells (NPCs) into mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in rapid rejection that is mediated, in part, by T cells. However, the contribution of the innate immune response to allograft rejection in a model of viral-induced neurological disease has not been well defined. Herein, we demonstrate that the natural killer (NK) cell-expressing-activating receptor NKG2D participates in transplanted allogeneic NPC rejection in mice persistently infected with JHMV. Cultured NPCs derived from C57BL/6 (H-2(b) ) mice express the NKG2D ligand retinoic acid early precursor transcript (RAE)-1 but expression was dramatically reduced upon differentiation into either glia or neurons. RAE-1(+) NPCs were susceptible to NK cell-mediated killing whereas RAE-1(-) cells were resistant to lysis. Transplantation of C57BL/6-derived NPCs into JHMV-infected BALB/c (H-2(d) ) mice resulted in infiltration of NKG2D(+) CD49b(+) NK cells and treatment with blocking antibody specific for NKG2D increased survival of allogeneic NPCs. Furthermore, transplantation of differentiated RAE-1(-) allogeneic NPCs into JHMV-infected BALB/c mice resulted in enhanced survival, highlighting a role for the NKG2D/RAE-1 signaling axis in allograft rejection. We also demonstrate that transplantation of allogeneic NPCs into JHMV-infected mice resulted in infection of the transplanted cells suggesting that these cells may be targets for infection. Viral infection of cultured cells increased RAE-1 expression, resulting in enhanced NK cell-mediated killing through NKG2D recognition. Collectively, these results show that in a viral-induced demyelination model, NK cells contribute to rejection of allogeneic NPCs through an NKG2D signaling pathway. © 2014 AlphaMed Press.

  7. Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

    PubMed Central

    Mao, Mao; Zhou, Liang

    2017-01-01

    We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases. PMID:28194184

  8. 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1alpha,25(OH)2D3 in hypertrophic cells.

    PubMed

    Boyan, B D; Hurst-Kennedy, J; Denison, T A; Schwartz, Z

    2010-07-01

    Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH)2D3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1alpha,25(OH)2D3. 24R,25(OH)2D3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi). 24R,25(OH)2D3 blocked apoptotic effects in a dose-dependent manner. Similarly, apoptosis was induced in ATDC5 cell cultures and 24R,25(OH)2D3 blocked this effect. Further studies indicated that 24R,25(OH)2D3 acts via at least two independent pathways. 24R,25(OH)2D3 increases LPA receptor-1 (LPA R1) expression and production of lysophosphatidic acid (LPA), and subsequent LPA R1/3-dependent signaling, thereby decreasing p53 abundance. LPA also increases the Bcl-2/Bax ratio. In addition, 24R,25(OH)2D3 acts by increasing PKC activity. 24R,25(OH)2D3 stimulates 1-hydroxylase activity, resulting in increased levels of 1,25(OH)2D3, and it increases levels of phospholipase A2 activating protein, which is required for rapid 1alpha,25(OH)2D3-dependent activation of PKC in GC cells. These results suggest that 24R,25(OH)2D3 modulates growth plate development by controlling the rate and extent of RC chondrocyte transition to a GC chondrocyte phenotype.

  9. The relationship between terminal functionalization and molecular weight of a gene delivery polymer and transfection efficacy in mammary epithelial 2-D cultures and 3-D organotypic cultures

    PubMed Central

    Bhise, Nupura S.; Gray, Ryan S.; Sunshine, Joel C.; Htet, Soe; Ewald, Andrew J.

    2011-01-01

    Non-viral gene delivery vectors were developed for efficient gene transfer to hard to transfect mouse mammary epithelial cells. Ten modified versions of the same base poly(beta-amino ester), poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol), were tested in both traditional 2-D monolayer and in 3-D organotypic cultures. The polymers self-assembled with plasmid DNA encoding enhanced green fluorescent protein to form nanoparticles (~100 nm) used to transfect the cells. Nanoparticle transfection efficacy was tuned by changes in synthesis and fabrication conditions and the transfection efficacy was analyzed using confocal microscopy and flow cytometry. The best performing polymeric nanoparticles transfected 57±6% of the cells in 2-D culture and 6±1% of the cells in 3-D culture. Small modifications to the polymer end-capping molecules and tuning of polymer molecular weight could either significantly enhance the transfection efficacy up to 6-fold or instead abolish efficacy completely. The efficacy of leading polymers was higher than that of the commercial transfection agent FuGENE® HD by a factor of 13 in 2-D and 2 in 3-D. These non-viral nanoparticles may be useful as delivery reagents or targeted therapeutics for breast cancer. This gene delivery strategy is also a promising approach for studying the normal development of the mammary gland. PMID:20674001

  10. NKG2D functions as an activating receptor on natural killer cells in the common marmoset (Callithrix jacchus).

    PubMed

    Watanabe, Masamichi; Kudo, Yohei; Kawano, Mitsuko; Nakayama, Masafumi; Nakamura, Kyohei; Kameda, Mai; Ebara, Masamune; Sato, Takeki; Nakamura, Marina; Omine, Kaito; Kametani, Yoshie; Suzuki, Ryuji; Ogasawara, Kouetsu

    2014-11-01

    The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

  11. Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models

    PubMed Central

    Sung, Kyung Eun; Su, Xiaojing; Berthier, Erwin; Pehlke, Carolyn; Friedl, Andreas; Beebe, David J.

    2013-01-01

    The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models. PMID:24124550

  12. Unraveling the role of mechanical stimulation on smooth muscle cells: A comparative study between 2D and 3D models.

    PubMed

    Bono, N; Pezzoli, D; Levesque, L; Loy, C; Candiani, G; Fiore, G B; Mantovani, D

    2016-10-01

    A thorough understanding of cell response to combined culture configuration and mechanical cues is of paramount importance in vascular tissue engineering applications. Herein, we investigated and compared the response of vascular smooth muscle cells (vSMCs) cultured in different culture environments (2D cell monolayers and 3D cellularized collagen-based gels) in combination with mechanical stimulation (7% uniaxial cyclic strain, 1 Hz) for 2 and 5 days. When cyclic strain was applied, two different responses, in terms of cell orientation and expression of contractile-phenotype proteins, were observed in 2D and 3D models. Specifically, in 2D configuration, cyclic strain caused ∼50% of cell population to align nearly perpendicular (80-90 degrees) to the strain direction, while not influencing the contractile-phenotype protein expression, as compared to the 2D static controls. Conversely, the application of uniaxial strain to 3D constructs induced a ∼60% cell alignment almost parallel (0-10 degrees) to the strain direction. Moreover, 3D mechanical stimulation applied for 5 days induced a twofold increase of SM α-actin level and a 14-fold increase of calponin expression as compared to 3D static controls. Altogether these findings provide a new insight into the potential to drive cell behavior by modulating the extracellular matrix and the biomechanical environment. Biotechnol. Bioeng. 2016;113: 2254-2263. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. NK cells enhance dendritic cell response against parasite antigens via NKG2D pathway.

    PubMed

    Guan, Hongbing; Moretto, Magali; Bzik, David J; Gigley, Jason; Khan, Imtiaz A

    2007-07-01

    Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional, the mechanism or molecules involved in this cross-talk have not been identified. In this study, we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8+ T cell response by these cells. In vitro blockade of NKG2D, a molecule present on human and murine NK cells, neutralizes the NK cell-induced up-regulation of DC response. Moreover, treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8+ T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction, which apparently is important for the generation of robust CD8+ T cell immunity against intracellular pathogens. To the best of our knowledge, this is the first work that describes in vivo importance of NKG2D during natural infection.

  14. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

    This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

  15. Common Effects on Cancer Cells Exerted by a Random Positioning Machine and a 2D Clinostat

    PubMed Central

    Svejgaard, Benjamin; Wehland, Markus; Ma, Xiao; Kopp, Sascha; Sahana, Jayashree; Warnke, Elisabeth; Aleshcheva, Ganna; Hemmersbach, Ruth; Hauslage, Jens; Grosse, Jirka; Bauer, Johann; Corydon, Thomas Juhl; Islam, Tawhidul; Infanger, Manfred; Grimm, Daniela

    2015-01-01

    In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML-1; RO82-W-1), which were exposed to a 2D clinostat (CLINO), a random positioning machine (RPM) and to normal 1g-conditions. After a three (3d)- or seven-day-culture (7d) on the two devices, we found both cell types growing three-dimensionally within multicellular spheroids (MCS) and also cells remaining adherent (AD) to the culture flask, while 1g-control cultures only formed adherent monolayers, unless the bottom of the culture dish was covered by agarose. In this case, the cytokines IL-6 and IL-8 facilitated the formation of MCS in both cell lines using the liquid-overlay technique at 1g. ML-1 cells grown on the RPM or the CLINO released amounts of IL-6 and MCP-1 into the supernatant, which were significantly elevated as compared to 1g-controls. Release of IL-4, IL-7, IL-8, IL-17, eotaxin-1 and VEGF increased time-dependently, but was not significantly influenced by the gravity conditions. After 3d on the RPM or the CLINO, an accumulation of F-actin around the cellular membrane was detectable in AD cells of both cell lines. IL-6 and IL-8 stimulation of ML-1 cells for 3d and 7d influenced the protein contents of ß1-integrin, talin-1, Ki-67, and beta-actin dose-dependently in adherent cells. The ß1-integrin content was significantly decreased in AD and MCS samples compared with 1g, while talin-1 was higher expressed in MCS than AD populations. The proliferation marker Ki-67 was elevated in AD samples compared with 1g and MCS samples. The ß-actin content of R082-W-1 cells remained unchanged. ML-1 cells exhibited no change in ß-actin in RPM cultures, but a reduction in CLINO samples. Thus, we concluded that simulated microgravity influences the release of cytokines in follicular thyroid cancer cells, and the production of ß1-integrin and talin-1 and predicts an identical effect under real microgravity conditions. PMID:26274317

  16. Common Effects on Cancer Cells Exerted by a Random Positioning Machine and a 2D Clinostat.

    PubMed

    Svejgaard, Benjamin; Wehland, Markus; Ma, Xiao; Kopp, Sascha; Sahana, Jayashree; Warnke, Elisabeth; Aleshcheva, Ganna; Hemmersbach, Ruth; Hauslage, Jens; Grosse, Jirka; Bauer, Johann; Corydon, Thomas Juhl; Islam, Tawhidul; Infanger, Manfred; Grimm, Daniela

    2015-01-01

    In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML-1; RO82-W-1), which were exposed to a 2D clinostat (CLINO), a random positioning machine (RPM) and to normal 1g-conditions. After a three (3d)- or seven-day-culture (7d) on the two devices, we found both cell types growing three-dimensionally within multicellular spheroids (MCS) and also cells remaining adherent (AD) to the culture flask, while 1g-control cultures only formed adherent monolayers, unless the bottom of the culture dish was covered by agarose. In this case, the cytokines IL-6 and IL-8 facilitated the formation of MCS in both cell lines using the liquid-overlay technique at 1g. ML-1 cells grown on the RPM or the CLINO released amounts of IL-6 and MCP-1 into the supernatant, which were significantly elevated as compared to 1g-controls. Release of IL-4, IL-7, IL-8, IL-17, eotaxin-1 and VEGF increased time-dependently, but was not significantly influenced by the gravity conditions. After 3d on the RPM or the CLINO, an accumulation of F-actin around the cellular membrane was detectable in AD cells of both cell lines. IL-6 and IL-8 stimulation of ML-1 cells for 3d and 7d influenced the protein contents of ß1-integrin, talin-1, Ki-67, and beta-actin dose-dependently in adherent cells. The ß1-integrin content was significantly decreased in AD and MCS samples compared with 1g, while talin-1 was higher expressed in MCS than AD populations. The proliferation marker Ki-67 was elevated in AD samples compared with 1g and MCS samples. The ß-actin content of R082-W-1 cells remained unchanged. ML-1 cells exhibited no change in ß-actin in RPM cultures, but a reduction in CLINO samples. Thus, we concluded that simulated microgravity influences the release of cytokines in follicular thyroid cancer cells, and the production of ß1-integrin and talin-1 and predicts an identical effect under real microgravity conditions.

  17. An omics approach to rational feed: Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics.

    PubMed

    Blondeel, Eric J M; Ho, Raymond; Schulze, Steffen; Sokolenko, Stanislav; Guillemette, Simon R; Slivac, Igor; Durocher, Yves; Guillemette, J Guy; McConkey, Brendan J; Chang, David; Aucoin, Marc G

    2016-09-20

    Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis

    PubMed Central

    Zhang, Jiyuan; Dominguez-Sola, David; Hussein, Shafinaz; Lee, Ji-Eun; Holmes, Antony B.; Bansal, Mukesh; Vlasevska, Sofija; Mo, Tongwei; Tang, Hongyan; Basso, Katia; Ge, Kai; Dalla-Favera, Riccardo; Pasqualucci, Laura

    2015-01-01

    Mutations in the gene encoding the KMT2D (also called MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of KMT2D mutations and their role in lymphomagenesis are unknown. Here we show that FL/DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B-cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B-cells and enhances B cell proliferation in mice. In mice overexpressing BCL2, which develop GC-derived lymphomas resembling human tumors, genetic ablation of Kmt2d leads to a further increase in tumor incidence. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may represent a rational therapeutic approach for targeting early tumorigenic events. PMID:26366712

  19. T Cells Infiltrating Diseased Liver Express Ligands for the NKG2D Stress Surveillance System

    PubMed Central

    Huang, Wei-Chen; Easom, Nicholas J.; Tang, Xin-Zi; Gill, Upkar S.; Singh, Harsimran; Robertson, Francis; Chang, Chiwen; Trowsdale, John; Davidson, Brian R.; Rosenberg, William M.; Fusai, Giuseppe; Toubert, Antoine; Kennedy, Patrick T.; Peppa, Dimitra

    2017-01-01

    NK cells, which are highly enriched in the liver, are potent regulators of antiviral T cells and immunopathology in persistent viral infection. We investigated the role of the NKG2D axis in T cell/NK cell interactions in hepatitis B. Activated and hepatitis B virus (HBV)–specific T cells, particularly the CD4 fraction, expressed NKG2D ligands (NKG2DL), which were not found on T cells from healthy controls (p < 0.001). NKG2DL-expressing T cells were strikingly enriched within HBV-infected livers compared with the periphery or to healthy livers (p < 0.001). NKG2D+NK cells were also increased and preferentially activated in the HBV-infected liver (p < 0.001), in direct proportion to the percentage of MICA/B-expressing CD4 T cells colocated within freshly isolated liver tissue (p < 0.001). This suggests that NKG2DL induced on T cells within a diseased organ can calibrate NKG2D-dependent activation of local NK cells; furthermore, NKG2D blockade could rescue HBV-specific and MICA/B-expressing T cells from HBV-infected livers. To our knowledge, this is the first ex vivo demonstration that non-virally infected human T cells can express NKG2DL, with implications for stress surveillance by the large number of NKG2D-expressing NK cells sequestered in the liver. PMID:28031333

  20. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    PubMed Central

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-01-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work. PMID:28262671

  1. Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells.

    PubMed

    Flynn, Maxfield P; Fiedler, Sarah E; Karlsson, Amelia B; Carr, Daniel W; Maizels, Evelyn T; Hunzicker-Dunn, Mary

    2016-08-01

    Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and β-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation.

  2. Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells

    PubMed Central

    2016-01-01

    ABSTRACT Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and β-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation. PMID:27335427

  3. NKG2D receptor regulates human effector T-cell cytokine production

    PubMed Central

    Barber, Amorette

    2011-01-01

    Although innate immune signals shape the activation of naive T cells, it is unclear how innate signals influence effector T-cell function. This study determined the effects of stimulating the NKG2D receptor in conjunction with the TCR on human effector CD8+ T cells. Stimulation of CD8+ T cells through CD3 and NKG2D simultaneously or through a chimeric NKG2D receptor, which consists of NKG2D fused to the intracellular region of CD3ζ, activated β-catenin and increased expression of β-catenin–induced genes, whereas T cells stimulated through the TCR or a combination of the TCR and CD28 did not. Activation by TCR and NKG2D prevented expression and production of anti-inflammatory cytokines IL-10, IL-9, IL-13, and VEGF-α in a β-catenin– and PPARγ- dependent manner. NKG2D stimulation also modulated the cytokine secretion of T cells activated simultaneously through CD3 and CD28. These data indicate that activating CD8+ T cells through the NKG2D receptor along with the TCR modulates signal transduction and the production of anti-inflammatory cytokines. Thus, human effector T cells alter their function depending on which innate receptors are engaged in conjunction with the TCR complex. PMID:21518928

  4. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection

    PubMed Central

    Deng, Weiwen; Gowen, Benjamin G.; Zhang, Li; Wang, Lin; Lau, Stephanie; Iannello, Alexandre; Xu, Jianfeng; Rovis, Tihana L.; Xiong, Na; Raulet, David H.

    2016-01-01

    Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, shedding of MULT1, a high affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are inhibitory, and suggest a new approach for cancer immunotherapy. PMID:25745066

  5. CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells

    PubMed Central

    Sauer, M; Schuldner, M; Hoffmann, N; Cetintas, A; Reiners, K S; Shatnyeva, O; Hallek, M; Hansen, H P; Gasser, S; von Strandmann, E P

    2017-01-01

    Tumor surveillance of natural killer (NK) cells is mediated by the cytotoxicity receptor natural-killer group 2 member D (NKG2D). Ligands for NKG2D are generally not expressed on healthy cells, but induced on the surface of malignant cells. To date, NKG2D ligand (NKG2D-L) induction was mainly described to depend on the activation of the DNA damage response, although the molecular mechanisms that regulate NKG2D-L expression remain largely unknown. Here, we show that the acetyltransferases CBP (CREB-binding protein) and p300 play a crucial role in the regulation of NKG2D-L on tumor cells. Loss of CBP/p300 decreased the basal cell surface expression of human ligands and reduced the upregulation of MICA/B and ULBP2 in response to histone deacetylase inhibitors or DNA damage. Furthermore, CBP/P300 deficiency abrogated the sensitivity of stressed cells to NK cell-mediated killing. CBP/p300 were also identified as major regulators of mouse NKG2D ligand RAE-1 in vitro and in vivo using the Eμ-Myc lymphoma model. Mechanistically, we observed an enhanced activation of the CBP/p300 binding transcription factor CREB (cAMP response element-binding protein) correlating to the NKG2D-L upregulation. Moreover, increased binding of CREB and CBP/p300 to NKG2D-L promoters and elevated histone acetylation were detectable. This study provides strong evidence for a major role of CBP and p300 in orchestrating NKG2D-L induction and consequently immunosurveillance of tumors in mice and humans. These findings might help to develop novel immunotherapeutic approaches against cancer. PMID:27477692

  6. Crossing barriers: the new dimension of 2D cell migration assays.

    PubMed

    Van Horssen, Remco; ten Hagen, Timo L M

    2011-01-01

    In our body cells move in three dimensions, embedded in an extracellular matrix that varies in composition, density and stiffness, and this movement is fundamental to life. Next to 3D cell migration assays, representing these physiological circumstances, still we need 2D migrations assays to perform detailed studies on the contribution of matrix-components and (extra)cellular proteins to cell movements. Next to the debate on differences between 3D and 2D migration, there also are many new perspectives on the use and development of novel or modified 2D cell migration assays. Of special significance is the introduction of so-called barrier migration assays, methods that avoid cell and matrix damage, as complementation or replacement of scratch/wound healing assays. Here, we discuss the possibilities and limitations of different 2D barrier migration assays.

  7. Silibinin affects tumor cell growth because of reduction of stemness properties and induction of apoptosis in 2D and 3D models of MDA-MB-468.

    PubMed

    Abdollahi, Pegah; Ebrahimi, Marzieh; Motamed, Nasrin; Samani, Fazel S

    2015-06-01

    Silibinin, with a strong antioxidant activity and a weak cytotoxic property, is considered a candidate for cancer prevention. As there is no information on its effect on breast cancer tumor-initiating cells [cancer stem cells (CSCs)] in a 3D culture model, which more closely mimic natural tissues, we carried out this study to determine whether silibinin can target breast CSCs in MDA-MB-468 cells cultured under 3D and 2D conditions. Silibinin was added to culture medium of MDA-MB-468 at a half maximal inhibitory concentration (IC50) dose in 2D and 3D models. Then, stemness properties were assessed using colony and sphere-formation tests. Flow cytometry and real-time PCR were used to determine the different expression levels of stem cell-related marker at protein and mRNA levels under both culture conditions. Our results showed that silibinin inhibits cell growth in a dose-dependent manner by induction of apoptosis, alteration of the cell cycle, reduction of stemness properties and function, and induction of tumoral differentiation. The mechanism of silibinin action and also the response of tumor cells differed when cells were cultured in a 3D model compared with a 2D model. Silibinin may potentially target breast CSCs. Moreover, tumor-initiating cells are more sensitive to silibinin in a 3D culture than in a 2D culture.

  8. Loss of MEF2D expression inhibits differentiation and contributes to oncogenesis in rhabdomyosarcoma cells.

    PubMed

    Zhang, Meiling; Truscott, Jamie; Davie, Judith

    2013-11-27

    Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the defects present in these cells that block myogenesis. Protein and RNA analysis identified the loss of MEF2D in RMS cells. MEF2D was expressed in RD and RH30 cells by transient transfection and selection of stable cell lines, respectively, to demonstrate the rescue of muscle differentiation observed. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were used with RH30 cells expressing MEF2D to demonstrate the loss of oncogenic growth in vitro and xenograft assays were used to confirm the loss of tumor growth in vivo. Here, we show that one member of the MEF2 family of proteins required for normal myogenesis, MEF2D, is largely absent in RMS cell lines representing both major subtypes of RMS as well as primary cells derived from an embryonal RMS model. We show that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find that MyoD and myogenin are bound with their dimerization partner, the E proteins, to the promoters of muscle specific genes in RMS cells. However, we cannot detect MEF2D binding at any promoter tested. We find that exogenous MEF2D expression can activate muscle specific luciferase constructs, up regulate p21 expression and increase muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility and anchorage independent growth in vitro. We have confirmed the inhibition of tumorigenicity by MEF2D in a tumor xenograft model, with a complete regression of tumor growth. Our data indicate that the oncogenic properties of RMS cells can be partially attributed to the loss of MEF2D expression and

  9. Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.

    PubMed

    Meng, Xiaoting; Li, Wenfei; Young, Fraser; Gao, Runchi; Chalmers, Laura; Zhao, Min; Song, Bing

    2012-02-16

    Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system(1,2). These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans(3,4). In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types(5,6), including neural progenitor cells (NPCs)(7,8). Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously(5,11). Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures(9,10). Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

  10. Human NKG2D-ligands: cell biology strategies to ensure immune recognition

    PubMed Central

    Fernández-Messina, Lola; Reyburn, Hugh T.; Valés-Gómez, Mar

    2012-01-01

    Immune recognition mediated by the activating receptor NKG2D plays an important role for the elimination of stressed cells, including tumors and virus-infected cells. On the other hand, the ligands for NKG2D can also be shed into the sera of cancer patients where they weaken the immune response by downmodulating the receptor on effector cells, mainly NK and T cells. Although both families of NKG2D-ligands, major histocompatibility complex class I-related chain (MIC) A/B and UL16 binding proteins (ULBPs), are related to MHC molecules and their expression is increased after stress, many differences are observed in terms of their biochemical properties and cell trafficking. In this paper, we summarize the variety of NKG2D-ligands and propose that selection pressure has driven evolution of diversity in their trafficking and shedding, but not receptor binding affinity. However, it is also possible to identify functional properties common to individual ULBP molecules and MICA/B alleles, but not generally conserved within the MIC or ULBP families. These characteristics likely represent examples of convergent evolution for efficient immune recognition, but are also attractive targets for pathogen immune evasion strategies. Categorization of NKG2D-ligands according to their biological features, rather than their genetic family, may help to achieve a better understanding of NKG2D-ligand association with disease. PMID:23056001

  11. NKG2D Signaling Leads to NK Cell Mediated Lysis of Childhood AML

    PubMed Central

    Ditthard, Kerstin; Lang, Peter; Mezger, Markus; Michaelis, Sebastian; Handgretinger, Rupert

    2015-01-01

    Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression. PMID:26236752

  12. Divergences in KIR2D+ natural killer and KIR2D+CD8+ T-cell reconstitution following liver transplantation.

    PubMed

    López-Álvarez, M R; Campillo, J A; Legaz, I; Blanco-García, R M; Salgado-Cecilia, G; Bolarín, J M; Gimeno, L; Gil, J; García-Alonso, A M; Muro, M; Alvarez-López, M R; Miras, M; Minguela, A

    2011-03-01

    Natural killer (NK) and CD8(+) T cells may be active elements in the allograft response, but little is known about their role in liver transplantation. Some of these cells express killer immunoglobulin-like receptors (KIRs), which after binding specific ligands may transmit inhibitory/activating signals. In this study, circulating NK and CD8(+) T cells expressing CD158a/h (KIR2DL1/S1) or CD158b/j (KIR2DL2/3/S(2)) receptors were analyzed in 142 liver recipients by flow cytometry. They were underrepresented in patients before transplantation, but following transplantation, whereas the KIR2D(+) NK subsets experienced a late recuperation (day 365) mainly in C2-homozygous patients developing early acute rejection, recovery of the 2 CD8(+)KIR2D(+) T cells started earlier, showing significant differences on day 365 between patients without acute rejection and those suffering from it (p = 0.004 and p < 0.0001, respectively). These differences were also evident when the human leukocute antigen-C genotypes of the recipient were considered. In conclusion, whereas the late recovery of KIR2D(+) NK cells in C2/C2 patients appears to be linked to acute rejection, the increase in early CD8(+)KIR2D(+) T cells in overall liver recipients correlates with a most successful early graft outcome. Therefore, monitoring of KIR2D(+) cells appears to be a useful tool for liver transplant follow-up.

  13. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    NASA Astrophysics Data System (ADS)

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  14. NKG2D+ IFN-γ+ CD8+ T Cells Are Responsible for Palladium Allergy

    PubMed Central

    Kawano, Mitsuko; Nakayama, Masafumi; Aoshima, Yusuke; Nakamura, Kyohei; Ono, Mizuho; Nishiya, Tadashi; Nakamura, Syou; Takeda, Yuri; Dobashi, Akira; Takahashi, Akiko; Endo, Misato; Ito, Akiyo; Ueda, Kyosuke; Sato, Naoki; Higuchi, Shigehito; Kondo, Takeru; Hashimoto, Suguru; Watanabe, Masamichi; Watanabe, Makoto; Takahashi, Tetsu; Sasaki, Keiichi; Nakamura, Masanori; Sasazuki, Takehiko; Narushima, Takayuki; Suzuki, Ryuji; Ogasawara, Kouetsu

    2014-01-01

    Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8+ T cells are responsible for the disease as CD8+ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8+ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8+ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D+ CD8+ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy. PMID:24533050

  15. Naringenin enhances NK cell lysis activity by increasing the expression of NKG2D ligands on Burkitt's lymphoma cells.

    PubMed

    Kim, Jeong Hwa; Lee, Jae Kwon

    2015-11-01

    Natural killer (NK) cells are capable of identifying and killing tumor cells as well as virus infected cells without pre-sensitization. NK cells express activating and inhibitory receptors, and can distinguish between normal and tumor cells. The present study was designed to demonstrate the importance of the expression level of NKG2D ligands on the Burkitt's lymphoma cell line, Raji, in enhancing NK cell cytolytic activity. Various flavonoids were used as stimulants to enhance the expression of NKG2D ligands. NK cell lysis activity against Raji was not changed by pre-treatment of Raji with luteolin, kaempferol, taxifolin and hesperetin. However, treatment of Raji with naringenin showed increased sensitivity to NK cell lysis than untreated control cells. The activity of naringenin was due to enhanced NKG2D ligand expression. These results provide evidence that narigenin's antitumor activity may be due to targeting of NKG2D ligand expression and suggests a possible immunotherapeutic role for cancer treatment.

  16. Natural Killer Group 2, Member D/NKG2D Ligands in Hematopoietic Cell Transplantation

    PubMed Central

    Carapito, Raphael; Aouadi, Ismail; Ilias, Wassila; Bahram, Seiamak

    2017-01-01

    Natural killer group 2, member D (NKG2D) is an invariant activatory receptor present on subsets of natural killer and T lymphocytes. It stimulates the cytolytic effector response upon engagement of its various stress-induced ligands NKG2D ligands (NKG2DL). Malignant transformation and conditioning treatment prior to hematopoietic cell transplantation (HCT) are stress factors leading to the activation of the NKG2D/NKG2DL signaling in clinical settings. In the context of HCT, NKG2D-bearing cells can kill both tumor and healthy cells expressing NKG2DL. The NKG2D/NKG2DL engagement has therefore a key role in the regulation of one of the most salient issues in allogeneic HCT, i.e., maintaining a balance between graft-vs.-leukemia effect and graft-vs.-host disease. The present review summarizes the current state of our knowledge pertaining to the role of the NKG2D and NKG2DL in HCT. PMID:28396673

  17. Radiolocalization of human small cell lung cancer and antigen-positive normal tissues using monoclonal antibody LS2D617

    SciTech Connect

    Wilson, B.S.; Petrella, E.; Lowe, S.R.; Lien, K.; Mackensen, D.G.; Gridley, D.S.; Stickney, D.R. )

    1990-05-15

    The murine monoclonal antibody LS2D617, which reacts with an antigen associated with human small cell lung carcinoma (SCLC), was tested in preclinical models to assess its potential for specific targeting of tumors in human SCLC cancer patients. LS2D617 detects a cell antigen on the surface of cultured SCLC and neuroblastoma cell lines. Scatchard analysis of the binding of LS2D617 to NCIH69 SCLC cells indicates an affinity constant of about 1 x 10(8) M-1 and an epitope expression level of approximately 2 x 10(6) antigenic sites/cell. Molecular weight analysis of the target antigen and antibody competition experiments showed that LS2D617 should be classified as a SCLC Cluster 1 antibody. LS2D617 was labeled with 111In and tested for biodistribution (4, 24, 48, 72, and 96 h postinjection) in nude mice bearing the human SCLC NCIH69 tumor. Tumor values peaked at about 35% injected dose/g (Day 3) compared with about 8% injected dose/g for an irrelevant IgG1 antibody while normal tissue accumulation for both antibodies was about 2-8% injected dose/g. Immunohistochemical studies demonstrated that LS2D617 reacts with the central nervous system, peripheral nerves, endocrine tissues, and heart tissue of rabbits as it does in human tissues. The ability of LS2D617 to accumulate in vivo in normal tissues that express the specific target antigen was tested in rabbits. Rabbits given i.v. injections of 111In-LS2D617 or control labeled antibody were sacrificed at 48 h and tissues were examined by gamma well counting, autoradiography, and immunohistochemical staining for murine immunoglobulin. Specific uptake was seen in all sites defined as antigen positive by immunohistology (i.e., heart, liver bile duct, peripheral nerves, pituitary, adrenal), except the central nervous system (brain and spinal cord) which was inaccessible to antibody because of the blood brain barrier.

  18. The NKG2D ligand ULBP4 binds to TCRgamma9/delta2 and induces cytotoxicity to tumor cells through both TCRgammadelta and NKG2D.

    PubMed

    Kong, Yan; Cao, Wei; Xi, Xueyan; Ma, Chi; Cui, Lianxian; He, Wei

    2009-07-09

    UL16-binding proteins (ULBPs) belong to a family of ligands for NKG2D activating receptor of human natural killer (NK) cells. We previously reported that RAET1E2, a soluble isoform of the RAET1E (ULBP4), inhibits NKG2D-mediated NK cytotoxicity. In this study, we examined whether ULBP4 could be recognized by gammadeltaT cells via TCRgammadelta. Here we show that immobilized soluble ULBP4 (rULBP4) induces the proliferation of human ovarian epithelial carcinoma- or colonic carcinoma-derived Vdelta2(+) T cells in vitro. These Vdelta2(+) T cells secrete Th1 cytokines and display a strong cytolytic activity toward ULBP4-transfected targets. We also show that ULBP4 binds to a soluble chimeric protein containing TCRgamma9/delta2 and activates TCR(-) Jurkat T cells transfected with TCRgamma9/delta2. Moreover, both TCRgammadelta and NKG2D are involved in ULBP4-induced activation and cytotoxicity of gammadeltaT cells. We found that ULBP4 is expressed not only on human tumor cells, but also on Epstein-Barr virus (EBV)-infected peripheral blood cells. Taken together, our data suggest that ULBP4 functions as a ligand for both TCRgammadelta and NKG2D and may play a key role in immune surveillance of tumor development and clearance of viral infection.

  19. Enhanced photon absorption in spiral nanostructured solar cells using layered 2D materials.

    PubMed

    Tahersima, Mohammad H; Sorger, Volker J

    2015-08-28

    Recent investigations of semiconducting two-dimensional (2D) transition metal dichalcogenides have provided evidence for strong light absorption relative to its thickness attributed to high density of states. Stacking a combination of metallic, insulating, and semiconducting 2D materials enables functional devices with atomic thicknesses. While photovoltaic cells based on 2D materials have been demonstrated, the reported absorption is still just a few percent of the incident light due to their sub-wavelength thickness leading to low cell efficiencies. Here we show that taking advantage of the mechanical flexibility of 2D materials by rolling a molybdenum disulfide (MoS(2))/graphene (Gr)/hexagonal boron nitride stack to a spiral solar cell allows for optical absorption up to 90%. The optical absorption of a 1 μm long hetero-material spiral cell consisting of the aforementioned hetero stack is about 50% stronger compared to a planar MoS(2) cell of the same thickness; although the volumetric absorbing material ratio is only 6%. A core-shell structure exhibits enhanced absorption and pronounced absorption peaks with respect to a spiral structure without metallic contacts. We anticipate these results to provide guidance for photonic structures that take advantage of the unique properties of 2D materials in solar energy conversion applications.

  20. Enhanced photon absorption in spiral nanostructured solar cells using layered 2D materials

    NASA Astrophysics Data System (ADS)

    Tahersima, Mohammad H.; Sorger, Volker J.

    2015-08-01

    Recent investigations of semiconducting two-dimensional (2D) transition metal dichalcogenides have provided evidence for strong light absorption relative to its thickness attributed to high density of states. Stacking a combination of metallic, insulating, and semiconducting 2D materials enables functional devices with atomic thicknesses. While photovoltaic cells based on 2D materials have been demonstrated, the reported absorption is still just a few percent of the incident light due to their sub-wavelength thickness leading to low cell efficiencies. Here we show that taking advantage of the mechanical flexibility of 2D materials by rolling a molybdenum disulfide (MoS2)/graphene (Gr)/hexagonal boron nitride stack to a spiral solar cell allows for optical absorption up to 90%. The optical absorption of a 1 μm long hetero-material spiral cell consisting of the aforementioned hetero stack is about 50% stronger compared to a planar MoS2 cell of the same thickness; although the volumetric absorbing material ratio is only 6%. A core-shell structure exhibits enhanced absorption and pronounced absorption peaks with respect to a spiral structure without metallic contacts. We anticipate these results to provide guidance for photonic structures that take advantage of the unique properties of 2D materials in solar energy conversion applications.

  1. Cell Counting in Human Endobronchial Biopsies - Disagreement of 2D versus 3D Morphometry

    PubMed Central

    Bratu, Vlad A.; Erpenbeck, Veit J.; Fehrenbach, Antonia; Rausch, Tanja; Rittinghausen, Susanne; Krug, Norbert; Hohlfeld, Jens M.; Fehrenbach, Heinz

    2014-01-01

    Question Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D) counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D) methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. Materials and Methods Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7) and smokers (n = 7), embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68) and T-lymphocytes (CD3), respectively. In identical fields of view, cell numbers per volume unit (NV) were assessed using the physical disector (3D), and profiles per area unit (NA) were counted (2D). For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. Results In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05). When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. Conclusions 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a ‘universal conversion formula’. PMID:24663339

  2. Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones.

    PubMed

    Drakulic, D; Krstic, A; Stevanovic, M

    2012-05-15

    SOX2, a universal marker of pluripotent stem cells, is a transcription factor that helps control embryonic development in vertebrates; its expression persists in neural stem/progenitor cells into adulthood. Considering the critical role of the SOX2 transcription factor in the regulation of genes required for self-renewal and pluripotency of stem cells, we developed and characterized SOX2-overexpressing NT2/D1 cell clones. Using Southern blot and semi-quantitative RT-PCR, we confirmed integration and expression of exogenous SOX2 in three NT2/D1 cell clones. Overexpression of the SOX2 gene was detected in two of these clones. SOX2 overexpression in NT2/D1 cell clones resulted in altered expression of key pluripotency genes OCT4 and NANOG. Furthermore, SOX2-overexpressing NT2/D1 cell clones entered into retinoic acid-dependent neural differentiation, even when there was elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of neural markers (neuroD1 and synaptophysin) was higher in induced cell clones than in induced parental cells. The cell clone with SOX2 overexpression had an approximately 1.3-fold higher growth rate compared to parental cells. SOX2 overexpression did not increase the population of cells undergoing apoptosis. Taken together, we developed two SOX2-overexpressing cell clones, with constitutive SOX2 expression after three weeks of retinoic acid treatment. SOX2 overexpression resulted in altered expression of pluripotency-related genes, increased proliferation, and altered expression of neural markers after three weeks of retinoic acid treatment.

  3. Chimeric NKG2D CAR-Expressing T Cell-Mediated Attack of Human Ovarian Cancer Is Enhanced by Histone Deacetylase Inhibition

    PubMed Central

    Song, De-Gang; Ye, Qunrui; Santoro, Stephen; Fang, Chongyun; Best, Andrew

    2013-01-01

    Abstract NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4+ and CD8+ NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer. PMID:23297870

  4. Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition.

    PubMed

    Song, De-Gang; Ye, Qunrui; Santoro, Stephen; Fang, Chongyun; Best, Andrew; Powell, Daniel J

    2013-03-01

    NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4(+) and CD8(+) NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.

  5. The Effect of 1α,25(OH)2D3 on Osteogenic Differentiation of Stem Cells from Dental Pulp of Exfoliated Deciduous Teeth

    PubMed Central

    Mojarad, Farzad; Amiri, Iraj; Rafatjou, Rezvan; Janeshin, Atousa; Farhadian, Maryam

    2016-01-01

    Statement of the Problem: Stem cells from human exfoliated deciduous teeth (SHEDs) are a population of highly proliferative cells, being capable of differentiating into osteogenic, odontogenic, adipocytes, and neural cells. Vitamin D3 metabolites such as 1α, 25-dihydroxyvitamin D3 are key factors in the regulation of bone metabolism. Purpose: The aim of this study was to investigate the effect of 1α, 25-dihydroxyvitamin D3 on osteogenic differentiation (alkaline phosphatase activity and alizarin red staining) of stem cells of exfoliated deciduous teeth. Materials and Method: Dental pulp was removed from freshly extracted primary teeth and immersed in a digestive solution. Then, the dental pulp cells were immersed in α-MEM (minimum essential medium) to which 10% fetal bovine serum was added. After the third passage, the cells were isolated from the culture plate and were used for osteogenic differentiation. As a control group, the cells were cultured in osteogenic cell culture medium. As the case group, the cells were cultured in osteogenic culture medium supplemented with 100 nM 1α,25 (OH)2D3. The alkaline phosphatase (ALP) activity and alizarin red staining were analyzed to evaluate the osteogenic differentiation at day 21. The results were analyzed by using t-test. Results: Compared with the control group, significant increase was observed in ALP activity of SHEDs after being treated with 1α,25(OH)2D3 (p= 0.002). Alizarin red staining demonstrated that the cells exposed to 1α,25(OH)2D3 induced higher mineralized nodules (p< 0.001). Conclusion: Osteoblast differentiation in SHEDs was stimulated by 1α,25(OH) 2D3. It can be concluded that 1α,25(OH)2D3 can improve osteoblastic differentiation. PMID:27942551

  6. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    PubMed

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-02-28

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  7. Multiwell capillarity-based microfluidic device for the study of 3D tumour tissue-2D endothelium interactions and drug screening in co-culture models.

    PubMed

    Virumbrales-Muñoz, María; Ayuso, José María; Olave, Marta; Monge, Rosa; de Miguel, Diego; Martínez-Lostao, Luis; Le Gac, Séverine; Doblare, Manuel; Ochoa, Ignacio; Fernandez, Luis J

    2017-09-20

    The tumour microenvironment is very complex, and essential in tumour development and drug resistance. The endothelium is critical in the tumour microenvironment: it provides nutrients and oxygen to the tumour and is essential for systemic drug delivery. Therefore, we report a simple, user-friendly microfluidic device for co-culture of a 3D breast tumour model and a 2D endothelium model for cross-talk and drug delivery studies. First, we demonstrated the endothelium was functional, whereas the tumour model exhibited in vivo features, e.g., oxygen gradients and preferential proliferation of cells with better access to nutrients and oxygen. Next, we observed the endothelium structure lost its integrity in the co-culture. Following this, we evaluated two drug formulations of TRAIL (TNF-related apoptosis inducing ligand): soluble and anchored to a LUV (large unilamellar vesicle). Both diffused through the endothelium, LUV-TRAIL being more efficient in killing tumour cells, showing no effect on the integrity of endothelium. Overall, we have developed a simple capillary force-based microfluidic device for 2D and 3D cell co-cultures. Our device allows high-throughput approaches, patterning different cell types and generating gradients without specialised equipment. We anticipate this microfluidic device will facilitate drug screening in a relevant microenvironment thanks to its simple, effective and user-friendly operation.

  8. Adaptation of a 2-D Clinostat for Simulated Microgravity Experiments with Adherent Cells

    NASA Astrophysics Data System (ADS)

    Eiermann, Peter; Kopp, Sascha; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

    2013-10-01

    The fast-rotating 2-D clinostat, a ground-based facility for investigations in simulated microgravity, is mainly used for experiments with cell suspensions. Here, we describe the adaptation of a 2-D clinostat for adherent cell investigations using commercially available slide flasks. As a gradient of residual accelerations is present in the slide flasks during clinorotation, the range of maximal g-values has to be adjusted to the investigated cells and type of analysis. For gene expression analysis, a harvesting slide chamber was constructed, allowing collection of cells exposed to defined g-values. Using this slide chamber, human 1F6 melanoma cell line, exposed in the ranges of ≤0.012 g, ≤0.024 g, or ≤0.036 g for 24 h, was harvested and the respective mRNA levels of guanylyl cyclase A (GC-A), an enzyme catalyzing cyclic GMP synthesis, were determined by real-time quantitative PCR analysis. Our results show that the down-regulation of GC-A mRNA levels in 1F6 melanoma cells depends on the residual acceleration values with a maximal reduction at ≤0.012 g. We further used the slide flasks by the clinorotation of murine RAW 264.7 macrophage cell line for f-actin analysis. The laser scanning microscopy images of cells exposed to g-values of ≤0.006 g for 1 h show an increase in the cell size of clinorotated cells, but no rearrangement in the f-actin filament system compared to static 1-g controls. Thus, 2-D clinostats equipped with slide flasks can be used for adherent cell experiments, however, the maximal g-values have to be carefully considered.

  9. The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

    PubMed Central

    Isernhagen, Antje; Malzahn, Dörthe; Viktorova, Elena; Elsner, Leslie; Monecke, Sebastian; von Bonin, Frederike; Kilisch, Markus; Wermuth, Janne Marieke; Walther, Neele; Balavarca, Yesilda; Stahl-Hennig, Christiane; Engelke, Michael; Walter, Lutz; Bickeböller, Heike; Kube, Dieter; Wulf, Gerald; Dressel, Ralf

    2015-01-01

    The MHC class I chain-related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)-cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA-129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft-versus-host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA-129Val/Val genotype carriers was improved when treated with anti-thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA-129Met isoform was characterized by stronger NKG2D signaling, triggering more NK-cell cytotoxicity and interferon-γ release, and faster co-stimulation of CD8+ T cells. The MICA-129Met variant also induced a faster and stronger down-regulation of NKG2D on NK and CD8+ T cells than the MICA-129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA-129Met variants appeared to reduce the severity of aGVHD. PMID:26483398

  10. Spheroid Culture of Mesenchymal Stem Cells

    PubMed Central

    Cesarz, Zoe; Tamama, Kenichi

    2016-01-01

    Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown. PMID:26649054

  11. Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition.

    PubMed

    Di Modica, Martina; Sfondrini, Lucia; Regondi, Viola; Varchetta, Stefania; Oliviero, Barbara; Mariani, Gabriella; Bianchi, Giulia Valeria; Generali, Daniele; Balsari, Andrea; Triulzi, Tiziana; Tagliabue, Elda

    2016-01-05

    Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.

  12. A 2-D Piston Effect Solution for the Relaxation in the MISTE Cell

    NASA Technical Reports Server (NTRS)

    Weilert, Mark

    2003-01-01

    When a large density stratification is no longer a problem in a microgravity environment, one would like to increase the sample size in order to increase the signal-to-noise ratio for a specific heat measurement. To reduce the equilibration time associated with the large sample size, we designed a cylindrical cell containing a stack of plates that separate the bulk fluid into 60 equally thin layers. To understand the thermal behavior of the whole cell, we analyzed the thermal behavior of a 2-D composite system of a cylindrical near-critical fluid layer in contact with a cylindrical copper plate. In this 2-D analysis, the circumference boundary of the two cylindrical layers is subjected to a step temperature change. The solution of this 2-D composite system includes the piston effect that speeds up the equilibration in the near-critical fluid layer and the pure diffusion in the copper plate. The results of this analysis indicate that the characteristic length for the equilibration of the stacked cell is determined by an effective thickness of a single fluid layer instead of the total height of the cylindrical cell.

  13. RAE-1 ligands for the NKG2D receptor are regulated by E2F transcription factors, which control cell cycle entry

    PubMed Central

    Jung, Heiyoun; Hsiung, Benjamin; Pestal, Kathleen; Procyk, Emily

    2012-01-01

    The NKG2D stimulatory receptor expressed by natural killer cells and T cell subsets recognizes cell surface ligands that are induced on transformed and infected cells and facilitate immune rejection of tumor cells. We demonstrate that expression of retinoic acid early inducible gene 1 (RAE-1) family NKG2D ligands in cancer cell lines and proliferating normal cells is coupled directly to cell cycle regulation. Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry. Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D. Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses. These findings suggest that cellular proliferation, as occurs in cancer cells but also other pathological conditions, is a key signal tied to immune reactions mediated by NKG2D-bearing lymphocytes. PMID:23166357

  14. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  15. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  16. Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

    PubMed Central

    Jensen, Helle; Folkersen, Lasse; Skov, Søren

    2012-01-01

    NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

  17. Computational efficient segmentation of cell nuclei in 2D and 3D fluorescent micrographs

    NASA Astrophysics Data System (ADS)

    De Vylder, Jonas; Philips, Wilfried

    2011-02-01

    This paper proposes a new segmentation technique developed for the segmentation of cell nuclei in both 2D and 3D fluorescent micrographs. The proposed method can deal with both blurred edges as with touching nuclei. Using a dual scan line algorithm its both memory as computational efficient, making it interesting for the analysis of images coming from high throughput systems or the analysis of 3D microscopic images. Experiments show good results, i.e. recall of over 0.98.

  18. Proteomic analysis using 2-D liquid separations of intact proteins from whole-cell lysates.

    PubMed

    Zhu, Kan; Yan, Fang; O'Neil, Kimberly A; Hamler, Rick; Lubman, David M; Lin, Linda; Barder, Timothy J

    2004-02-01

    This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.

  19. Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

    PubMed

    Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

    2012-01-01

    Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

  20. Effects of NKG2D haplotypes on the cell-surface expression of NKG2D protein on natural killer and CD8 T cells of peripheral blood among atomic-bomb survivors.

    PubMed

    Imai, Kazue; Hayashi, Tomonori; Yamaoka, Mika; Kajimura, Junko; Yoshida, Kengo; Kusunoki, Yoichiro; Nakachi, Kei

    2012-06-01

    NKG2D is a primary activating receptor that triggers cell-mediated cytotoxicity in NK cells against tumor and virus-infected cells. We previously identified the NKG2D haplotypes in the natural killer gene complex region on chromosome 12p. Two major haplotype alleles, LNK1 and HNK1, were closely related to low and high natural cytotoxic activity phenotypes, respectively. Furthermore, the haplotype of HNK1/HNK1 has revealed a decreased risk of cancer compared with LNK1/LNK1. In the present study, using flow cytometry, we evaluated the functional effects of NKG2D haplotypes and five htSNPs in terms of the cell-surface expression of NKG2D protein on NK and CD8 T cells of peripheral blood among 732 atomic-bomb survivors. NKG2D expression on NK cells showed significant increases, in the order of LNK1/LNK1, LNK1/HNK1 and HNK1/HNK1 haplotypes (p for trend=0.003), or with major homozygous, heterozygous, and minor homozygous genotypes for individual htSNPs (p for trend=0.02-0.003). The same trend was observed for NKG2D expression on CD8 T cells. Our findings indicate that the NKG2D haplotypes are associated with the expression levels of NKG2D protein on NK and CD8 T cells, resulting in inter-individual variations in human cytotoxic response.

  1. Fas-ligand-mediated paracrine T cell regulation by the receptor NKG2D in tumor immunity.

    PubMed

    Groh, Veronika; Smythe, Kimberly; Dai, Zhenpeng; Spies, Thomas

    2006-07-01

    Tumor-associated ligands of the activating NKG2D receptor can effectively stimulate T cell responses at early but not late stages of tumor growth. In late-stage human tumor settings, we observed MIC-driven proliferation of NKG2D(+)CD4(+) T cells that produced the cytokine Fas ligand (FasL) as a result of NKG2D costimulation but were themselves protected from Fas-mediated growth arrest. In contrast, FasL suppressed proliferation of T cells in vitro that did not receive NKG2D costimulation. Similar observations with normal peripheral blood NKG2D(+)CD8(+) T cells demonstrated unrecognized NKG2D-mediated immune functions, whereby FasL release promotes tumor cell death and NKG2D costimulation prolongs T cell survival. These effects, beneficial in conditions of limited NKG2D ligand expression, may be counterweighed when massive expression and shedding of MIC occurs, such as in some late-stage tumors, that causes sustained NKG2D costimulation and population expansion of immunosuppressive T cells.

  2. 2D light scattering static cytometry for label-free single cell analysis with submicron resolution.

    PubMed

    Xie, Linyan; Yang, Yan; Sun, Xuming; Qiao, Xu; Liu, Qiao; Song, Kun; Kong, Beihua; Su, Xuantao

    2015-11-01

    Conventional optical cytometric techniques usually measure fluorescence or scattering signals at fixed angles from flowing cells in a liquid stream. Here we develop a novel cytometer that employs a scanning optical fiber to illuminate single static cells on a glass slide, which requires neither microfluidic fabrication nor flow control. This static cytometric technique measures two dimensional (2D) light scattering patterns via a small numerical aperture (0.25) microscope objective for label-free single cell analysis. Good agreement is obtained between the yeast cell experimental and Mie theory simulated patterns. It is demonstrated that the static cytometer with a microscope objective of a low resolution around 1.30 μm has the potential to perform high resolution analysis on yeast cells with distributed sizes. The capability of the static cytometer for size determination with submicron resolution is validated via measurements on standard microspheres with mean diameters of 3.87 and 4.19 μm. Our 2D light scattering static cytometric technique may provide an easy-to-use, label-free, and flow-free method for single cell diagnostics. © 2015 International Society for Advancement of Cytometry.

  3. Role of 2-D periodic symmetrical nanostructures in improving efficiency of thin film solar cells

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Jiang, Liyong; Li, Xiangyin

    2016-01-01

    We systematically investigated several different nanostructures in crystalline silicon (c-Si) thin film solar cells and then proposed a brand-new structure with two dimensional (2-D) periodic dielectric cylinders on the top and annular metal columns on bottom surface to enhance the optical harvesting. The periodic symmetrical nanostructures affect the solar cell efficiency due to the grating diffraction effect of dielectric columns and surface plasmon polaritons (SPPs) effect induced by metal nanostructures at the dielectric-metal interface. About 52.1% more optical absorption and 33.3% more power conversion efficiency are obtained, and the maximum short current reaches to 33.24 mA/cm2.

  4. 2D-CELL: image processing software for extraction and analysis of 2-dimensional cellular structures

    NASA Astrophysics Data System (ADS)

    Righetti, F.; Telley, H.; Leibling, Th. M.; Mocellin, A.

    1992-01-01

    2D-CELL is a software package for the processing and analyzing of photographic images of cellular structures in a largely interactive way. Starting from a binary digitized image, the programs extract the line network (skeleton) of the structure and determine the graph representation that best models it. Provision is made for manually correcting defects such as incorrect node positions or dangling bonds. Then a suitable algorithm retrieves polygonal contours which define individual cells — local boundary curvatures are neglected for simplicity. Using elementary analytical geometry relations, a range of metric and topological parameters describing the population are then computed, organized into statistical distributions and graphically displayed.

  5. 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.

  6. 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.

  7. 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.

  8. PURE CULTURES OF CELLS

    PubMed Central

    Carrel, Alexis

    1912-01-01

    In experiment I a group of ameboid cells was isolated from a culture of cardiac muscle sixty-three days old, and cultivated in plasma. After several passages, they formed a dense tissue from which ameboid cells radiated. The culture was divided into two parts. The part cultivated in plasma alone kept its morphological characters and continued to produce ameboid cells. The part cultivated upon silk in plasma became modified; the cells lost their ameboid characters, and were transformed into large elongated cells which were united in chains, or interlaced to form a network. In experiment II the round cells taken from a culture of connective tissue seventy-four days old multiplied rapidly. They transformed themselves into elongated cells and produced, after a few passages, a mass of dense connective tissue. From the tissue a large number of elongated cells were constantly growing. In both experiments the tissues originated from the ameboid or round cells extirpated from cultures that were sixty-three and seventy-four days old respectively. These cultures were still growing actively thirty and forty days later; that is, more than one hundred days after the extirpation of the original fragments from the organism. These experiments show that from old cultures it is possible to isolate and propagate cells that belong to a definite type. A tissue, formed by a pure strain of cells, can be obtained in this way, and this new method may be of value in cytological investigations. PMID:19867562

  9. Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy

    PubMed Central

    Ghasemi, Reza; Lazear, Eric; Wang, Xiaoli; Arefanian, Saeed; Zheleznyak, Alexander; Carreno, Beatriz M.; Higashikubo, Ryuji; Gelman, Andrew E.; Kreisel, Daniel; Fremont, Daved H.; Krupnick, Alexander Sasha

    2016-01-01

    Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2Rα chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2Rα. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2Rα-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2. PMID:27650575

  10. Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy.

    PubMed

    Ghasemi, Reza; Lazear, Eric; Wang, Xiaoli; Arefanian, Saeed; Zheleznyak, Alexander; Carreno, Beatriz M; Higashikubo, Ryuji; Gelman, Andrew E; Kreisel, Daniel; Fremont, Daved H; Krupnick, Alexander Sasha

    2016-09-21

    Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2Rα chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2Rα. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2Rα-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2.

  11. 2D Homologous Perovskites as Light-Absorbing Materials for Solar Cell Applications.

    PubMed

    Cao, Duyen H; Stoumpos, Constantinos C; Farha, Omar K; Hupp, Joseph T; Kanatzidis, Mercouri G

    2015-06-24

    We report on the fabrication and properties of the semiconducting 2D (CH3(CH2)3NH3)2(CH3NH3)(n-1)Pb(n)I(3n+1) (n = 1, 2, 3, and 4) perovskite thin films. The band gaps of the series decrease with increasing n values, from 2.24 eV (CH3(CH2)3NH3)2PbI4 (n = 1) to 1.52 eV CH3NH3PbI3 (n = ∞). The compounds exhibit strong light absorption in the visible region, accompanied by strong photoluminescence at room temperature, rendering them promising light absorbers for photovoltaic applications. Moreover, we find that thin films of the semi-2D perovskites display an ultrahigh surface coverage as a result of the unusual film self-assembly that orients the [Pb(n)I(3n+1)](-) layers perpendicular to the substrates. We have successfully implemented this 2D perovskite family in solid-state solar cells, and obtained an initial power conversion efficiency of 4.02%, featuring an open-circuit voltage (V(oc)) of 929 mV and a short-circuit current density (J(sc)) of 9.42 mA/cm(2) from the n = 3 compound. This result is even more encouraging considering that the device retains its performance after long exposure to a high-humidity environment. Overall, the homologous 2D halide perovskites define a promising class of stable and efficient light-absorbing materials for solid-state photovoltaics and other applications.

  12. RAET1E2, a soluble isoform of the UL16-binding protein RAET1E produced by tumor cells, inhibits NKG2D-mediated NK cytotoxicity.

    PubMed

    Cao, Wei; Xi, Xueyan; Hao, Zhiyong; Li, Wenjing; Kong, Yan; Cui, Lianxian; Ma, Chi; Ba, Denian; He, Wei

    2007-06-29

    UL16-binding proteins (ULBPs, also termed as retinoic acid early transcripts, encoded by RAET1 genes), a family of ligands for NKG2D in humans, are frequently expressed by tumor cells and mediate cytotoxicities of natural killer (NK) cells and CD8(+) alphabeta T cells to tumor cells. ULBP1, ULBP2, ULBP3, and RAET1L link to membrane through glycosylphosphatidylinositol, whereas RAET1E and RAET1G contain transmembrane and cytoplasmic domains. Proteolytic cleavage of ULBP2 produces truncated and soluble forms that may counteract NKG2D-mediated tumor immune surveillance. In this study, we report that RAET1E can produce a soluble, 35-kDa protein (termed as RAET1E2) lacking the transmembrane region by selective splicing in tumor cells. The expressions of both RAET1E2 transcripts and protein can be found in different tumor cells and tissues. Preincubation of NK-92 cells, a human NK cell line, with culture supernatants from tumor cell lines expressing RAET1E2 or RAET1E2 gene-transfected COS-7 cells resulted in decreased expression of NKG2D on NK-92 cells. Furthermore, incubation of NK-92 cells with recombinant RAET1E2 protein also decreased the surface expression of NKG2D and resulted in marked reduction in cytotoxicities to MGC-803, HepG2, or K562 tumor cells. Taken together, our data provide strong evidence for an immune escape mechanism of tumors via alternative splicing of ULBP RNA to generate a free soluble ULBP protein, RAET1E2, that may impair NKG2D-mediated NK cell cytotoxicity to tumors.

  13. One-Year stable perovskite solar cells by 2D/3D interface engineering.

    PubMed

    Grancini, G; Roldán-Carmona, C; Zimmermann, I; Mosconi, E; Lee, X; Martineau, D; Narbey, S; Oswald, F; De Angelis, F; Graetzel, M; Nazeeruddin, Mohammad Khaja

    2017-06-01

    Despite the impressive photovoltaic performances with power conversion efficiency beyond 22%, perovskite solar cells are poorly stable under operation, failing by far the market requirements. Various technological approaches have been proposed to overcome the instability problem, which, while delivering appreciable incremental improvements, are still far from a market-proof solution. Here we show one-year stable perovskite devices by engineering an ultra-stable 2D/3D (HOOC(CH2)4NH3)2PbI4/CH3NH3PbI3 perovskite junction. The 2D/3D forms an exceptional gradually-organized multi-dimensional interface that yields up to 12.9% efficiency in a carbon-based architecture, and 14.6% in standard mesoporous solar cells. To demonstrate the up-scale potential of our technology, we fabricate 10 × 10 cm(2) solar modules by a fully printable industrial-scale process, delivering 11.2% efficiency stable for >10,000 h with zero loss in performances measured under controlled standard conditions. This innovative stable and low-cost architecture will enable the timely commercialization of perovskite solar cells.

  14. One-Year stable perovskite solar cells by 2D/3D interface engineering

    NASA Astrophysics Data System (ADS)

    Grancini, G.; Roldán-Carmona, C.; Zimmermann, I.; Mosconi, E.; Lee, X.; Martineau, D.; Narbey, S.; Oswald, F.; de Angelis, F.; Graetzel, M.; Nazeeruddin, Mohammad Khaja

    2017-06-01

    Despite the impressive photovoltaic performances with power conversion efficiency beyond 22%, perovskite solar cells are poorly stable under operation, failing by far the market requirements. Various technological approaches have been proposed to overcome the instability problem, which, while delivering appreciable incremental improvements, are still far from a market-proof solution. Here we show one-year stable perovskite devices by engineering an ultra-stable 2D/3D (HOOC(CH2)4NH3)2PbI4/CH3NH3PbI3 perovskite junction. The 2D/3D forms an exceptional gradually-organized multi-dimensional interface that yields up to 12.9% efficiency in a carbon-based architecture, and 14.6% in standard mesoporous solar cells. To demonstrate the up-scale potential of our technology, we fabricate 10 × 10 cm2 solar modules by a fully printable industrial-scale process, delivering 11.2% efficiency stable for >10,000 h with zero loss in performances measured under controlled standard conditions. This innovative stable and low-cost architecture will enable the timely commercialization of perovskite solar cells.

  15. One-Year stable perovskite solar cells by 2D/3D interface engineering

    PubMed Central

    Grancini, G.; Roldán-Carmona, C.; Zimmermann, I.; Mosconi, E.; Lee, X.; Martineau, D.; Narbey, S.; Oswald, F.; De Angelis, F.; Graetzel, M.; Nazeeruddin, Mohammad Khaja

    2017-01-01

    Despite the impressive photovoltaic performances with power conversion efficiency beyond 22%, perovskite solar cells are poorly stable under operation, failing by far the market requirements. Various technological approaches have been proposed to overcome the instability problem, which, while delivering appreciable incremental improvements, are still far from a market-proof solution. Here we show one-year stable perovskite devices by engineering an ultra-stable 2D/3D (HOOC(CH2)4NH3)2PbI4/CH3NH3PbI3 perovskite junction. The 2D/3D forms an exceptional gradually-organized multi-dimensional interface that yields up to 12.9% efficiency in a carbon-based architecture, and 14.6% in standard mesoporous solar cells. To demonstrate the up-scale potential of our technology, we fabricate 10 × 10 cm2 solar modules by a fully printable industrial-scale process, delivering 11.2% efficiency stable for >10,000 h with zero loss in performances measured under controlled standard conditions. This innovative stable and low-cost architecture will enable the timely commercialization of perovskite solar cells. PMID:28569749

  16. Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses.

    PubMed

    Watanabe, Naohide; Lam, Eric

    2011-06-01

    Cysteine proteases such as caspases play important roles in programmed cell death (PCD) of metazoans. Plant metacaspases (MCPs), a family of cysteine proteases structurally related to caspases, have been hypothesized to be ancestors of metazoan caspases, despite their different substrate specificity. Arabidopsis thaliana contains six type II MCP genes (AtMCP2a-f). Whether and how these individual members are involved in controlling PCD in plants remains largely unknown. Here we investigated the function and regulation of AtMCP2d, the predominant and constitutively expressed member of type II MCPs, in stress-inducible PCD. Two AtMCP2d mutants (mcp2d-1 and mcp2d-3) exhibited reduced sensitivity to PCD-inducing mycotoxin fumonisin B1 as well as oxidative stress inducers, whereas AtMCP2d over-expressors were more sensitive to these agents, and exhibited accelerated cell-death progression. We found that AtMCP2d exclusively localizes to the cytosol, and its accumulation and self-processing patterns were age-dependent in leaves. Importantly, active proteolytic processing of AtMCP2d proteins dependent on its catalytic activity was observed in mature leaves during mycotoxin-induced cell death. We also found that mcp2d-1 leaves exhibited reduced cell death in response to Pseudomonas syringae carrying avirulent gene avrRpt2, and that self-processing of AtMCP2d was also detected in wild-type leaves in response to this pathogen. Furthermore, increases in processed AtMCP2d proteins were found to correlate with conditional cell-death induction in two lesion-mimic mutants (cpr22 and ssi4) that exhibit spontaneous cell-death phenotypes. Taken together, our data strongly suggest that AtMCP2d plays a positive regulatory role in biotic and abiotic stress-induced PCD.

  17. Studies on the antigenicity of the NKG2D ligand H60a in tumour cells

    PubMed Central

    Yadav, Deepak; Ngolab, Jennifer; Dang, Natalie; Bui, Jack D

    2011-01-01

    H60a is a minor histocompatibility antigen expressed in BALB and 129/Sv but not C57BL/6 mouse strains. The majority of CD8+ T cells in C57BL/6 mice responding to BALB.B splenocytes are specific for H60a. Interestingly, H60a is expressed constitutively on tumour cells, but its nature as a tumour rejection antigen, as a parallel to its function as a transplant rejection antigen, has not been studied. In this report, we show that tumour cells that constitutively express H60a at the cell surface can be recognized by H60a-specific T cells. Furthermore, when H60a-expressing sarcoma cell lines are transplanted into C57BL/6 mice, H60a-specific T cells can be found at high percentages among the tumour-infiltrating CD8+ T cells. These findings were seen in C57BL/6 but not F1 (C57BL/6 × 129) mice (which express H60a), suggesting that endogenous tolerance mechanisms suppress the antigenic properties of H60a. Our findings have implications for the generation of tumour vaccines against human natural killer group 2D ligands, such as MHC class I chain-like gene A, that are also transplantation antigens. PMID:21438873

  18. Analysis of a 2D simulation model of biofilms with autonomous cells

    NASA Astrophysics Data System (ADS)

    Bhandar, Anand; Tatek, Yergou; Slater, Gary

    2006-03-01

    Biofilms are substances consisting of a large number of microorganisms that grow on surfaces in contact with liquids. They can be found growing in water pipes, on surgical instruments or on tooth surfaces. Mathematical models have been used for the last three decades in order to improve our understanding of their growth and behavior. We have designed and implemented a new Monte Carlo model based on the life of autonomous cells and investigated the static and dynamic characteristics of the resulting bacterial populations. Each cell is modeled as an autonomous agent whose behavior is controlled by thermodynamic parameters, mechanical properties, physiological rules and environmental conditions. In the 2D version studied, a cell is represented by a closed chain of self-avoiding beads linked together using the bond fluctuation algorithm. The cell is controlled both by the rigidity of its membrane and a pressure difference. The model is complemented by key features such as the processes of cell division, growth and death, attractive interactions between the cell and the surface, and the explicit presence of nutrient diffusion. Tuning model parameters leads to the growth and maturation of various types of biofilms. Typical colonies incorporating these and other important characteristics of biofilms such as the exopolymeric substance (EPS), metabolism and waste production, cell motility and chemotaxy, and cell mutation will be presented.

  19. Negative regulation of NKG2D expression by IL-4 in memory CD8 T cells.

    PubMed

    Ventre, Erwan; Brinza, Lilia; Schicklin, Stephane; Mafille, Julien; Coupet, Charles-Antoine; Marçais, Antoine; Djebali, Sophia; Jubin, Virginie; Walzer, Thierry; Marvel, Jacqueline

    2012-10-01

    IL-4 is one of the main cytokines produced during Th2-inducing pathologies. This cytokine has been shown to affect a number of immune processes such as Th differentiation and innate immune responses. However, the impact of IL-4 on CD8 T cell responses remains unclear. In this study, we analyzed the effects of IL-4 on global gene expression profiles of Ag-induced memory CD8 T cells in the mouse. Gene ontology analysis of this signature revealed that IL-4 regulated most importantly genes associated with immune responses. Moreover, this IL-4 signature overlapped with the set of genes preferentially expressed by memory CD8 T cells over naive CD8 T cells. In particular, IL-4 downregulated in vitro and in vivo in a STAT6-dependent manner the memory-specific expression of NKG2D, thereby increasing the activation threshold of memory CD8 T cells. Furthermore, IL-4 impaired activation of memory cells as well as their differentiation into effector cells. This phenomenon could have an important clinical relevance as patients affected by Th2 pathologies such as parasitic infections or atopic dermatitis often suffer from viral-induced complications possibly linked to inefficient CD8 T cell responses.

  20. Antitumor immunity. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection.

    PubMed

    Deng, Weiwen; Gowen, Benjamin G; Zhang, Li; Wang, Lin; Lau, Stephanie; Iannello, Alexandre; Xu, Jianfeng; Rovis, Tihana L; Xiong, Na; Raulet, David H

    2015-04-03

    Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D-activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, a shed form of MULT1, a high-affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are always inhibitory and suggest a new approach for cancer immunotherapy. Copyright © 2015, American Association for the Advancement of Science.

  1. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  2. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  3. SOX2 overexpression affects neural differentiation of human pluripotent NT2/D1 cells.

    PubMed

    Klajn, A; Drakulic, D; Tosic, M; Pavkovic, Z; Schwirtlich, M; Stevanovic, M

    2014-11-01

    SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics.

  4. Enhanced durability of polymer electrolyte membrane fuel cells by functionalized 2D boron nitride nanoflakes.

    PubMed

    Oh, Keun-Hwan; Lee, Dongju; Choo, Min-Ju; Park, Kwang Hyun; Jeon, Seokwoo; Hong, Soon Hyung; Park, Jung-Ki; Choi, Jang Wook

    2014-05-28

    We report boron nitride nanoflakes (BNNFs), for the first time, as a nanofiller for polymer electrolyte membranes in fuel cells. Utilizing the intrinsic mechanical strength of two-dimensional (2D) BN, addition of BNNFs even at a marginal content (0.3 wt %) significantly improves mechanical stability of the most representative hydrocarbon-type (HC-type) polymer electrolyte membrane, namely sulfonated poly(ether ether ketone) (sPEEK), during substantial water uptake through repeated wet/dry cycles. For facile processing with BNNFs that frequently suffer from poor dispersion in most organic solvents, we non-covalently functionalized BNNFs with 1-pyrenesulfonic acid (PSA). Besides good dispersion, PSA supports efficient proton transport through its sulfonic functional groups. Compared to bare sPEEK, the composite membrane containing BNNF nanofiller exhibited far improved long-term durability originating from enhanced dimensional stability and diminished chronic edge failure. This study suggests that introduction of properly functionalized 2D BNNFs is an effective strategy in making various HC-type membranes sustainable without sacrificing their original adventurous properties in polymer electrolyte membrane fuel cells.

  5. A 2-D enlarged cell technique (ECT) for elastic wave modelling on a curved free surface

    NASA Astrophysics Data System (ADS)

    Wei, Songlin; Zhuang, Mingwei; Zhou, Jianyang; Liu, Qing Huo

    2015-04-01

    The conventional finite-difference time-domain (FDTD) method for elastic waves suffers from the staircasing error when applied to model a curved free surface because of the structured grid. This is similar to the situation for the FDTD method in electromagnetics when it is applied to model a curved perfect conductor surface, where the conformal FDTD methods have been recently developed to avoid this error. In this work a stable and second-order accurate 2-D FDTD method for elastic wave modelling on a curved free surface is presented based on the finite volume method and enlarged cell technique (ECT). To achieve a sufficiently accurate implementation, a finite volume scheme is applied to the curved free surface to remove the staircasing error; in the meantime, to achieve the same stability as the FDTD method without reducing the time step increment, the ECT is introduced to preserve the solution stability even for small irregular cells. This method is verified by several 2-D numerical examples. Results show that the method is second-order accurate and stable at the Courant stability limit for a regular FDTD grid.

  6. Fish Stem Cell Cultures

    PubMed Central

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer. PMID:21547056

  7. Limbal melanocytes support limbal epithelial stem cells in 2D and 3D microenvironments.

    PubMed

    Dziasko, Marc A; Tuft, Stephen J; Daniels, Julie T

    2015-09-01

    Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the corneal epithelium of the ocular surface. LESCs are located within limbal crypts between the palisades of Vogt in the limbus; the interface between the peripheral cornea and conjunctiva. The limbal crypts have been proposed as a LESC niche owing to their support of epithelial cells, which can form holoclone colonies in vitro. Closely associated with the limbal crypts is a concentrated population of melanocytes. The anatomical location and close proximity to putative LESC suggests that melanocytes might play a role in maintenance of these stem cells in the niche. The aim of this study was to assess the ability of human limbal melanocytes (hLM) to support the expansion of human limbal epithelial cells (LECs) in vitro as an indicator of functional cell-cell interaction. After observing that hLM co-localize with clusters of compact epithelial cells in the native limbal crypts, hLM were isolated from crypt-rich cadaveric limbal biopsies and used as feeders for the culture of LECs. Interestingly, LECs grown on mitotically active hLM were able to generate large epithelial colonies that contained small and compact cells with morphological stem cell characteristics. Immunocytochemistry revealed that LECs expanded on hLM were positive for the expression of the putative stem cell markers CK15, Bmi-1 and p63α and negative for the marker of terminal cell differentiation CK3. LECs and hLM were finally co-cultured on RAFT (real architecture for 3D tissue) collagen tissue equivalents. In 3D co-cultures, hLM promoted multi-layering of the epithelial sheet in which basal cells were maintained in an undifferentiated state. Taken together, these observations suggest melanocytes could play an important role in the maintenance of LESCs in the native human limbal stem cell niche.

  8. Effects on Tumor Development and Metastatic Dissemination by the NKG2D Lymphocyte Receptor Expressed on Cancer Cells

    PubMed Central

    El-Gazzar, Ahmed; Cai, Xin; Reeves, Rebecca S.; Dai, Zhenpeng; Caballero-Benitez, Andrea; McDonald, David L.; Vazquez, Julio; Gooley, Ted A.; Sale, George E.; Spies, Thomas; Groh, Veronika

    2014-01-01

    The stimulatory NKG2D lymphocyte receptor together with its tumor-associated ligands enable the immune system to recognize and destroy cancer cells. However, with dynamic changes unfolding, cancers exploit NKG2D and its ligands for immune evasion and suppression. Recent findings have added yet another functional dimension wherein cancer cells themselves coopt NKG2D for their own benefit to complement the presence of its ligands for self stimulation of parameters of tumorigenesis. Those findings are here extended to in vivo tumorigenicity testing by employing orthotopic xenotransplant breast cancer models in mice. Using human cancer lines with ectopic NKG2D expression and RNAi-mediated protein depletion among other controls, we show that NKG2D self-stimulation has tumor promoting capacity. NKG2D signals had no notable effects on cancer cell proliferation and survival but acted at the level of angiogenesis, thus promoting tumor growth, tumor cell intravasation and dissemination. NKG2D-mediated effects on tumor initiation may represent another factor in the observed overall enhancement of tumor development. Altogether, these results may impact immunotherapy approaches, which currently do not account for such NKG2D effects in cancer patients and thus could be misdirected as underlying assumptions are incomplete. PMID:24141776

  9. Effects on tumor development and metastatic dissemination by the NKG2D lymphocyte receptor expressed on cancer cells.

    PubMed

    El-Gazzar, A; Cai, X; Reeves, R S; Dai, Z; Caballero-Benitez, A; McDonald, D L; Vazquez, J; Gooley, T A; Sale, G E; Spies, T; Groh, V

    2014-10-09

    The stimulatory NKG2D lymphocyte receptor together with its tumor-associated ligands enable the immune system to recognize and destroy cancer cells. However, with dynamic changes unfolding, cancers exploit NKG2D and its ligands for immune evasion and suppression. Recent findings have added yet another functional dimension, wherein cancer cells themselves co-opt NKG2D for their own benefit to complement the presence of its ligands for self-stimulation of parameters of tumorigenesis. Those findings are here extended to in vivo tumorigenicity testing by employing orthotopic xenotransplant breast cancer models in mice. Using human cancer lines with ectopic NKG2D expression and RNA interference (RNAi)-mediated protein depletion among other controls, we show that NKG2D self-stimulation has tumor-promoting capacity. NKG2D signals had no notable effects on cancer cell proliferation and survival but acted at the level of angiogenesis, thus promoting tumor growth, tumor cell intravasation and dissemination. NKG2D-mediated effects on tumor initiation may represent another factor in the observed overall enhancement of tumor development. Altogether, these results may have an impact on immunotherapy approaches, which currently do not account for such NKG2D effects in cancer patients and thus could be misdirected as underlying assumptions are incomplete.

  10. Changes in gene expression, protein content and morphology of chondrocytes cultured on a 3D Random Positioning Machine and 2D rotating clinostat

    NASA Astrophysics Data System (ADS)

    Aleshcheva, Ganna; Hauslage, Jens; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela; Sahana, Jayashree

    Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

  11. A 2-D Implicit, Energy and Charge Conserving Particle In Cell Method

    SciTech Connect

    McPherson, Allen L.; Knoll, Dana A.; Cieren, Emmanuel B.; Feltman, Nicolas; Leibs, Christopher A.; McCarthy, Colleen; Murthy, Karthik S.; Wang, Yijie

    2012-09-10

    Recently, a fully implicit electrostatic 1D charge- and energy-conserving particle-in-cell algorithm was proposed and implemented by Chen et al ([2],[3]). Central to the algorithm is an advanced particle pusher. Particles are moved using an energy conserving scheme and are forced to stop at cell faces to conserve charge. Moreover, a time estimator is used to control errors in momentum. Here we implement and extend this advanced particle pusher to include 2D and electromagnetic fields. Derivations of all modifications made are presented in full. Special consideration is taken to ensure easy coupling into the implicit moment based method proposed by Taitano et al [19]. Focus is then given to optimizing the presented particle pusher on emerging architectures. Two multicore implementations, and one GPU (Graphics Processing Unit) implementation are discussed and analyzed.

  12. 1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells

    PubMed Central

    Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N.; Glenn, Sean T.; Liu, Song; Trump, Donald L.; Johnson, Candace S.

    2014-01-01

    Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. MiRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253 J and 253J-BV cells express endogenous vitamin D receptor (VDR) which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253 J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. PMID:25263658

  13. Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis.

    PubMed

    Schrodt, Caroline; McHugh, Erin E; Gawinowicz, Mary Ann; Dupont, Herbert L; Brown, Eric L

    2013-01-01

    Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

  14. Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

    PubMed Central

    Schrodt, Caroline; McHugh, Erin E.; Gawinowicz, Mary Ann; DuPont, Herbert L.; Brown, Eric L.

    2013-01-01

    Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens. PMID:23922656

  15. Myocyte enhancer factor 2D regulates ectoderm specification and adhesion properties of animal cap cells in the early Xenopus embryo.

    PubMed

    Katz Imberman, Sandra; Kolpakova, Alina; Keren, Aviad; Bengal, Eyal

    2015-08-01

    In Xenopus, animal cap (AC) cells give rise to ectoderm and its derivatives: epidermis and the central nervous system. Ectoderm has long been considered a default pathway of embryonic development, with cells that are not under the influence of vegetal Nodal signaling adopting an ectodermal program of gene expression. In the present study, we describe the involvement of the animally-localized maternal transcription factor myocyte enhancer factor (Mef) 2D in regulating the identity of AC cells. We find that Mef2D is required for the formation of both ectodermal lineages: neural and epidermis. Gain and loss of function experiments indicate that Mef2D regulates early gastrula expression of key ectodermal/epidermal genes in the animal region. Mef2D controls the activity of zygotic bone morphogenetic protein (BMP) signaling known to dictate the epidermal differentiation program. Exogenous expression of Mef2D in vegetal blastomeres was sufficient to induce ectopic expression of ectoderm/epidermal genes in the vegetal half of the embryo, when Nodal signaling was inhibited. Depletion of Mef2D caused a loss of AC cell adhesion that was rescued by the expression of E-cadherin or bone morphogenetic protein 4. In addition, expression of Mef2D in the prospective endoderm caused unusual aggregation of vegetal cells with animal cells in vitro and inappropriate segregation to other germ layers in vivo. Mef2D cooperates with another animally-expressed transcription factor, FoxI1e. Together, they regulate the expression of genes encoding signaling proteins and the transcription factors that control the regional identity of animal cells. Therefore, we describe a new role for the animally-localized Mef2D protein in early ectoderm specification, which is similar to that of the vegetally-localized VegT in endoderm and mesoderm formation. © 2015 FEBS.

  16. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development

    PubMed Central

    Ortega-Molina, Ana; Boss, Isaac W.; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W.; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A.; Gascoyne, Randy D.; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M.; Wendel, Hans-Guido

    2015-01-01

    The lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center (GC) involution, impedes B cell differentiation and class switch recombination (CSR). Integrative genomic analyses indicate that KMT2D affects H3K4 methylation and expression of a specific set of genes including those in the CD40, JAK-STAT, Toll-like receptor, and B cell receptor pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3, and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell activating pathways. PMID:26366710

  17. Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

    PubMed Central

    Rajasekaran, Kamalakannan; Xiong, Va; Fong, Lee; Gorski, Jack; Malarkannan, Subramaniam

    2010-01-01

    Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8+ T cells. However, their independent functional contributions in distinct CD8+ T cell subsets are not well understood. In this study, CD8+ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA+CD27+) and memory (CD45RA−CD27+) CD8+ T cells (CD28Hi), while its expression was significantly lower in effector (CD45RA+CD27−) CD8+ T cells (CD28Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8+ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8+ T cells. Co-stimulation of CD28Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8+ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8+ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8+ T cells. However, boosting a recall immune response via memory CD8+ T cells or vaccination to stimulate naïve CD8+ T cells would require CD28-mediated co-stimulation. PMID:20844584

  18. Natural killer cell NKG2D and granzyme B are critical for allergic pulmonary inflammation⋆

    PubMed Central

    Farhadi, Nazanin; Lambert, Laura; Triulzi, Chiara; Openshaw, Peter J.M.; Guerra, Nadia; Culley, Fiona J.

    2014-01-01

    Background The diverse roles of innate immune cells in the pathogenesis of asthma remain to be fully defined. Natural killer (NK) cells are innate lymphocytes that can regulate adaptive immune responses. NK cells are activated in asthma; however, their role in allergic airway inflammation is not fully understood. Objective We investigated the importance of NK cells in house dust mite (HDM)-triggered allergic pulmonary inflammation. Specifically, we aimed to determine the role of the major NK-cell activating receptor NKG2D and NK-cell effector functions mediated by granzyme B. Methods Allergic airway inflammation was induced in the airways of mice by repeated intranasal HDM extract administration and responses in wild-type and NKG2D-deficient mice were compared. Adoptive transfer studies were used to identify the cells and mechanisms involved. Results Mice that lacked NKG2D were resistant to the induction of allergic inflammation and showed little pulmonary eosinophilia, few airway TH2 cells, and no rise in serum IgE after multiple HDM-allergen exposures. However, NKG2D was not required for pulmonary inflammation after a single inoculation of allergen. NKG2D-deficient mice showed no alteration in responses to respiratory virus infection. Transfer of wild-type NK cells (but not CD3+ cells) into NKG2D-deficient mice restored allergic inflammatory responses only if the NK cells expressed granzyme B. Conclusions These studies established a pivotal role for NK-cell NKG2D and granzyme B in the pathogenesis of HDM-induced allergic lung disease, and identified novel therapeutic targets for the prevention and treatment of asthma. PMID:24290277

  19. Optical and electrical study of organic solar cells with a 2D grating anode.

    PubMed

    Sha, Wei E I; Choy, Wallace C H; Wu, Yumao; Chew, Weng Cho

    2012-01-30

    We investigate both optical and electrical properties of organic solar cells (OSCs) incorporating 2D periodic metallic back grating as an anode. Using a unified finite-difference approach, the multiphysics modeling framework for plasmonic OSCs is established to seamlessly connect the photon absorption with carrier transport and collection by solving the Maxwell's equations and semiconductor equations (Poisson, continuity, and drift-diffusion equations). Due to the excited surface plasmon resonance, the significantly nonuniform and extremely high exciton generation rate near the metallic grating are strongly confirmed by our theoretical model. Remarkably, the nonuniform exciton generation indeed does not induce more recombination loss or smaller open-circuit voltage compared to 1D multilayer standard OSC device. The increased open-circuit voltage and reduced recombination loss by the plasmonic OSC are attributed to direct hole collections at the metallic grating anode with a short transport path. The work provides an important multiphysics understanding for plasmonic organic photovoltaics.

  20. Numerical Investigation of Penning Discharge Characteristics using 2D/3V Particle-In-Cell Method

    NASA Astrophysics Data System (ADS)

    Dikalyuk, A. S.; E Kuratov, S.

    2017-02-01

    Numerical model for the simulation of characteristics of Penning discharge is presented in the paper. The model is based on the 2D/3V axisymmetric electrostatic particle-in-cell algorithm implemented on unstructured triangular meshes. In order to simulate kinetic processes in the gas discharge plasma Monte-Carlo collision (MCC) method was incorporated into the model. Considered processes for the numerical experiments are elastic scattering of electrons on the neutral particles and ionization of neutral particles by electrons. Using this model various characteristics of Penning discharge such as anode and cathode currents, distribution of charged components in the discharge chamber have been calculated at various applied voltages and magnetic flux densities. The results of numerical experiments have been analyzed and discussed.

  1. Basic cell culture.

    PubMed

    Pollard, J W

    1990-01-01

    This article will describe the basic techniques required for successful cell culture. It will also act to introduce some of the other chapters in this volume. It is not intended, as this volume is not, to describe the establishment of a tissue culture laboratory, nor to provide a historical or theoretical survey of cell culture. There are several books that adequately cover these areas, including the now somewhat dated but still valuable volume by Paul (1), the multi-authored Methods in Enzymology volume edited by Jakoby and Pastan (2), and the new edition of Freshney (3). Instead, this chapter's focus will be on the techniques for establishing primary rodent cell cultures from embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of genetically pure strains. The cells described are all derived from Chinese hamsters since, to date, these cells, have proved to be the most useful for somatic cell genetics (4,5). The techniques, however, are generally applicable to most fibroblastic cell types.

  2. Effect of 1,25-(OH)2D3 and lipopolysaccharide on mononuclear cell inflammation in type 2 diabetes mellitus and diabetic nephropathy uremia.

    PubMed

    Wu, E L; Cui, H X

    2016-08-29

    The prevention and treatment of type-2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), which are disorders with high incidence rates, is of primary importance. In this study, we analyzed the effect of 1,25-(OH)2D3 and lipopolysaccharide (LPS) in combination with interleukin (IL)-15 on the inflammatory immune response and expression of vitamin D receptor (VDR) in mononuclear cells of T2DM and DN uremia (DNU) patients. The human acute monocytic leukemia cell line THP-1 was treated with peripheral blood serum isolated from 30 healthy controls and T2DM and DNU patients each, cultured in the presence or absence of 1,25-(OH)2D3, and subsequently treated with LPS and IL-15. The VDR mRNA and protein expression in THP-1 cells was detected by real-time polymerase chain reaction and western blot (and immunofluorescence assay), respectively, and IL-6 and IL-10 concentrations in the culture supernatant were detected by enzyme-linked immunosorbent assay. LPS treatment induced a significant decrease in VDR mRNA expression in T2DM and DNU serum-treated THP-1 cells compared to the control cells (P < 0.05). The VDR protein expression in DNU serum-treated THP-1 cells was also significantly down-regulated (P < 0.05). LPS treatment induced IL-6 secretion in serum-treated THP-1 cells (P < 0.05), while 1,25-(OH)2D3 treatment inhibited IL-6 secretion to some extent. These findings suggested that LPS down-regulates the expression of VDR in mononuclear cells of T2DM and DNU patients and induces an imbalance in the pro-inflammatory and anti-inflammatory cytokine response, while 1,25-(OH)2D3 partially reversed the effect of LPS and protected patients with T2DM and DNU.

  3. [Role of NKG2D-Expressing NK Cells and sMICA in Immune Surveillance of Advanced Lung Cancer.].

    PubMed

    Liang, Jing; Han, Fucai

    2009-01-20

    NKG2D-expressing NK cells and soluble major histocompatibility complex class I-related chain A (sMICA) is one of aroused general interests in tumor research area recently. The aim of the study is to investigate the levels of NKG2D-expressing NK cells and sMICA in peripheral blood of advanced lung cancer which are remarkably related to clinical significance and analyse the role of NKG2D-expressing NK cells and sMICA in immune surveillance. Flow cytometry was used to determine the percentage of NKG2D-expressing NK cells, T cell subsets, NK cells, and ELISA was used to mesure the levels of sMICA in peripheral blood of 115 advanced lung cancer patients and 50 healthy controls. Compared with control group, the levels of sMICA, CD8+T cells, NK cells increased, while the levels of NKG2D-expressing NK cells, CD3+ T cells, CD4+ T cells, CD4+ T/CD8+ T in experimental group decreased. NKG2D-expressing NK cells had a perfect negative correlation with sMICA (r =-0.319, P <0.05). NKG2D-expressing NK cells had positive correlation with CD4+ T cells, CD4+ T/CD8+ T and negative correlation with CD8+ T cells (P <0.05), sMICA had negative correlation with CD4+ T cells, CD4+ T/CD8+ T and positive correlation with CD8+ T cells (P <0.05), they had no significant correlation with CD3+ T cells, NK cells respectively (P <0.05). Accumulation of sMICA in serum may lead to the down-modulation of NKG2D-expressing NK which has been proposed to be a novel mechanism used by cancer cells to evade the tumor immunosurveillance. They may be potential indicators investigating immune functions and helpful in the evaluation of their happening and proceeding.

  4. PCaAnalyser: A 2D-Image Analysis Based Module for Effective Determination of Prostate Cancer Progression in 3D Culture

    PubMed Central

    Lovitt, Carrie J.; Avery, Vicky M.

    2013-01-01

    Three-dimensional (3D) in vitro cell based assays for Prostate Cancer (PCa) research are rapidly becoming the preferred alternative to that of conventional 2D monolayer cultures. 3D assays more precisely mimic the microenvironment found in vivo, and thus are ideally suited to evaluate compounds and their suitability for progression in the drug discovery pipeline. To achieve the desired high throughput needed for most screening programs, automated quantification of 3D cultures is required. Towards this end, this paper reports on the development of a prototype analysis module for an automated high-content-analysis (HCA) system, which allows for accurate and fast investigation of in vitro 3D cell culture models for PCa. The Java based program, which we have named PCaAnalyser, uses novel algorithms that allow accurate and rapid quantitation of protein expression in 3D cell culture. As currently configured, the PCaAnalyser can quantify a range of biological parameters including: nuclei-count, nuclei-spheroid membership prediction, various function based classification of peripheral and non-peripheral areas to measure expression of biomarkers and protein constituents known to be associated with PCa progression, as well as defining segregate cellular-objects effectively for a range of signal-to-noise ratios. In addition, PCaAnalyser architecture is highly flexible, operating as a single independent analysis, as well as in batch mode; essential for High-Throughput-Screening (HTS). Utilising the PCaAnalyser, accurate and rapid analysis in an automated high throughput manner is provided, and reproducible analysis of the distribution and intensity of well-established markers associated with PCa progression in a range of metastatic PCa cell-lines (DU145 and PC3) in a 3D model demonstrated. PMID:24278197

  5. PCaAnalyser: a 2D-image analysis based module for effective determination of prostate cancer progression in 3D culture.

    PubMed

    Hoque, Md Tamjidul; Windus, Louisa C E; Lovitt, Carrie J; Avery, Vicky M

    2013-01-01

    Three-dimensional (3D) in vitro cell based assays for Prostate Cancer (PCa) research are rapidly becoming the preferred alternative to that of conventional 2D monolayer cultures. 3D assays more precisely mimic the microenvironment found in vivo, and thus are ideally suited to evaluate compounds and their suitability for progression in the drug discovery pipeline. To achieve the desired high throughput needed for most screening programs, automated quantification of 3D cultures is required. Towards this end, this paper reports on the development of a prototype analysis module for an automated high-content-analysis (HCA) system, which allows for accurate and fast investigation of in vitro 3D cell culture models for PCa. The Java based program, which we have named PCaAnalyser, uses novel algorithms that allow accurate and rapid quantitation of protein expression in 3D cell culture. As currently configured, the PCaAnalyser can quantify a range of biological parameters including: nuclei-count, nuclei-spheroid membership prediction, various function based classification of peripheral and non-peripheral areas to measure expression of biomarkers and protein constituents known to be associated with PCa progression, as well as defining segregate cellular-objects effectively for a range of signal-to-noise ratios. In addition, PCaAnalyser architecture is highly flexible, operating as a single independent analysis, as well as in batch mode; essential for High-Throughput-Screening (HTS). Utilising the PCaAnalyser, accurate and rapid analysis in an automated high throughput manner is provided, and reproducible analysis of the distribution and intensity of well-established markers associated with PCa progression in a range of metastatic PCa cell-lines (DU145 and PC3) in a 3D model demonstrated.

  6. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  7. Autonomous Stimulation of Cancer Cell Plasticity by the Human NKG2D Lymphocyte Receptor Coexpressed with Its Ligands on Cancer Cells

    PubMed Central

    Cai, Xin; Dai, Zhenpeng; Reeves, Rebecca S.; Caballero-Benitez, Andrea; Duran, Kate L.; Delrow, Jeffrey J.; Porter, Peggy L.

    2014-01-01

    The stimulatory NKG2D receptor on lymphocytes promotes tumor immune surveillance by targeting ligands selectively induced on cancer cells. Progressing tumors counteract by employing tactics to disable lymphocyte NKG2D. This negative dynamic is escalated as some human cancer cells co-opt expression of NKG2D, thereby complementing the presence of its ligands for autonomous stimulation of oncogenic signaling. Clinical association data imply relationships between cancer cell NKG2D and metastatic disease. Here we show that NKG2D promotes cancer cell plasticity by induction of phenotypic, molecular, and functional signatures diagnostic of the epithelial–mesenchymal transition, and of stem-like traits via induction of Sox9, a key transcriptional regulator of breast stem cell maintenance. These findings obtained with model breast tumor lines and xenotransplants were recapitulated by ex vivo cancer cells from primary invasive breast carcinomas. Thus, NKG2D may have the capacity to drive high malignancy traits underlying metastatic disease. PMID:25291178

  8. Ataxia-telangiectasia mutated kinase-mediated upregulation of NKG2D ligands on leukemia cells by resveratrol results in enhanced natural killer cell susceptibility.

    PubMed

    Luis Espinoza, J; Takami, Akiyoshi; Trung, Ly Q; Nakao, Shinji

    2013-06-01

    The powerful activating receptor NKG2D is expressed by natural killer (NK) cells and promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). We report the effective induction of NKG2D-Ls, achieved with the naturally occurring polyphenol resveratrol, in a broad range of leukemia cells. In this study, resveratrol upregulated the NKG2D-Ls MHC class I chain-related proteins MICA and MICB, and UL16-binding proteins ULBP1, ULBP2, and ULBP3 in most of the leukemia cells analyzed. Ligand upregulation induced by resveratrol was impaired by pharmacological and genetic disruption of ataxia-telangiectasia mutated kinase, the main regulator of NKG2D-L expression. Leukemia cells treated with resveratrol were more susceptible to killing by NK cells than untreated cells, and the enhanced cytotoxicity of NK cells was blocked by treatment of NK cells with anti-NKG2D mAbs. Interestingly, resveratrol consistently upregulated the NKG2D receptor expression and enhanced NKG2D-mediated functions in resting NK cells obtained from healthy individuals. Therefore, resveratrol has attractive immunotherapeutic potential.

  9. Neuronal Cell Cultures.

    DTIC Science & Technology

    1982-10-01

    AD-A123 120 NEURONAL . CELL CULTURES( U) FEDERATION OF AMERICAN / SOCIETIES FOR EXPER IMENTAL B IOLOGY BETHES DA MD R BUNOF ET AL 01 0C 82 AFOSR-- a...REPRT NUM2. GOVT ACCESSION No. 3. RECIIENT’S CATALOG NUmBER 4. TITLE (end Subtitle) 5, TYPE OF REPORT & PERIOD COVERED Neuronal Cell Cultures FNLRPR...10. PROGRAM ELEMENT. PRZjECT. TASK~ Federation of American Societies for Experi- AREA & WORK UNIT N.jVBERS mental Biology (FASEB), 9650 Rockville

  10. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  11. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  12. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  13. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  14. Skeletal muscle satellite cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Molnar, Greg; Hartzell, Charles R.; Schroedl, Nancy A.; Gonda, Steve R.

    1993-01-01

    Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of

  15. Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.

    PubMed Central

    Deragon, J M; Sinnett, D; Labuda, D

    1990-01-01

    We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition. Images Fig. 2. Fig. 3. Fig. 4. PMID:1698616

  16. Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.

    PubMed

    Pal, Manoj; Moffa, Allison; Sreekumar, Arun; Ethier, Stephen P; Barder, Timothy J; Chinnaiyan, Arul; Lubman, David M

    2006-02-01

    A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxy-coated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunoblot analysis using anti-phosphotyrosine antibodies. A dynamic range of approximately 100 was achieved on the microarray when beta-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregulated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.

  17. 2D layered insulator hexagonal boron nitride enabled surface passivation in dye sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Shanmugam, Mariyappan; Jacobs-Gedrim, Robin; Durcan, Chris; Yu, Bin

    2013-10-01

    A two-dimensional layered insulator, hexagonal boron nitride (h-BN), is demonstrated as a new class of surface passivation materials in dye-sensitized solar cells (DSSCs) to reduce interfacial carrier recombination. We observe ~57% enhancement in the photo-conversion efficiency of the DSSC utilizing h-BN coated semiconductor TiO2 as compared with the device without surface passivation. The h-BN coated TiO2 is characterized by Raman spectroscopy to confirm the presence of highly crystalline, mixed monolayer/few-layer h-BN nanoflakes on the surface of TiO2. The passivation helps to minimize electron-hole recombination at the TiO2/dye/electrolyte interfaces. The DSSC with h-BN passivation exhibits significantly lower dark saturation current in the low forward bias region and higher saturation in the high forward bias region, respectively, suggesting that the interface quality is largely improved without impeding carrier transport at the material interface. The experimental results reveal that the emerging 2D layered insulator could be used for effective surface passivation in solar cell applications attributed to desirable material features such as high crystallinity and self-terminated/dangling-bond-free atomic planes as compared with high-k thin-film dielectrics.

  18. HLA class I, NKG2D, and natural cytotoxicity receptors regulate multiple myeloma cell recognition by natural killer cells.

    PubMed

    Carbone, Ennio; Neri, Paola; Mesuraca, Maria; Fulciniti, Mariateresa T; Otsuki, Takemi; Pende, Daniela; Groh, Veronika; Spies, Thomas; Pollio, Giuditta; Cosman, David; Catalano, Lucio; Tassone, Pierfrancesco; Rotoli, Bruno; Venuta, Salvatore

    2005-01-01

    The role of natural killer (NK) cells in multiple myeloma is not fully understood. Here, NK susceptibility of myeloma cells derived from distinct disease stages was evaluated in relation to major histocompatibility complex (MHC) class I, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), and UL16 binding protein (ULBP) expression. MHC class I molecules were hardly detectable on bone marrow cells of early-stage myeloma, while late-stage pleural effusion-derived cell lines showed a strong MHC class I expression. Conversely, a high MICA level was found on bone marrow myeloma cells, while it was low or not measurable on pleural effusion myeloma cells. The reciprocal surface expression of these molecules on bone marrow- and pleural effusion-derived cell was confirmed at mRNA levels. While bone marrow-derived myeloma cells were readily recognized by NK cells, pleural effusion-derived lines were resistant. NK protection of pleural effusion cells was MHC class I dependent. Receptor blocking experiments demonstrated that natural cytotoxicity receptor (NCR) and NK receptor member D of the lectin-like receptor family (NKG2D) were the key NK activating receptors for bone marrow-derived myeloma cell recognition. In ex vivo experiments patient's autologous fresh NK cells recognized bone marrow-derived myeloma cells. Our data support the hypothesis that NK cell cytotoxicity could sculpture myeloma and represents an important immune effector mechanism in controlling its intramedullary stages.

  19. Oscillating Cell Culture Bioreactor

    NASA Technical Reports Server (NTRS)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  20. 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size.

    PubMed

    Otani, Tomoki; Marchetto, Maria C; Gage, Fred H; Simons, Benjamin D; Livesey, Frederick J

    2016-04-07

    Variation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output.

  1. 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size

    PubMed Central

    Otani, Tomoki; Marchetto, Maria C.; Gage, Fred H.; Simons, Benjamin D.; Livesey, Frederick J.

    2016-01-01

    Summary Variation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output. PMID:27049876

  2. Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells.

    PubMed

    Ribiczey, Polett; Papp, Béla; Homolya, László; Enyedi, Ágnes; Kovács, Tünde

    2015-08-14

    We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3

  3. Characterization of DNAPL Source Zone Architecture and Associated Plume Response in 2-D Aquifer Cell Experiments

    NASA Astrophysics Data System (ADS)

    Granbery, E. K.; Cápiro, N.; Christ, J.; Pennell, K.

    2008-12-01

    A series of two-dimensional (2-D) aquifer cell experiments was conducted to evaluate the effects of DNAPL source zone architecture on down-gradient plume and flux-averaged effluent concentrations. The aquifer cells (1.0 m length x 48 cm height x 1.4 cm internal thickness) were packed with different size fractions of quartz sands and low permeability lens configurations to achieve a range of source zone saturation distributions. A mixed DNAPL, consisting of 1:1 trichloroethene (TCE) and tetrachloroethene (PCE), was released into the source zone region of the aquifer cell at a flow rate of 2.0 ml/min and allowed to redistribute for a period of 48 hours. The DNAPL saturation distribution was quantified using a light transmission system and characterized by the ganglia-to-pool ratio (GTP, 0 to infinity) and ganglia fraction (GF, 0-100%), where saturations less than 0.13 represent discrete ganglia and saturations equal to or greater than 0.13 are considered to represent "pools". Effluent dissolved-phase TCE and PCE concentrations were monitored continuously, while down-gradient plume concentrations were collected periodically from 25 side-port samples. To accelerate mass removal, the aquifer cells were flushed with 1.5 pore volumes of 4% Tween 80, a nonionic, food-grade surfactant that has been shown to facilitate microbial reductive dechlorination. Experimental data are used to evaluate interrelationships between the initial source zone architecture, mass removal, reductions in mass flux, and plume evolution.

  4. MIF-2/D-DT enhances proximal tubular cell regeneration through SLPI- and ATF4-dependent mechanisms.

    PubMed

    Ochi, Akinobu; Chen, Dong; Schulte, Wibke; Leng, Lin; Moeckel, Nickolas; Piecychna, Marta; Averdunk, Luisa; Stoppe, Christian; Bucala, Richard; Moeckel, Gilbert

    2017-09-01

    Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions that is produced by several organs and cell types. Depending on the target cell and the inflammatory context, MIF can engage its two component receptor complex CD74 and CD44 and the chemokine receptors CXCR2/4. MIF is constitutively expressed in renal proximal tubular cells, stored in intracellular preformed pools, and released at a low rate. Recently, a second MIF-like protein (i.e., MIF-2/D-DT) has been characterized in mammals. Our study was aimed at examining the role of MIF-2/D-DT, which mediates tissue protection in the heart, in tubular cell regeneration from ischemia-reperfusion injury. We found that Mif-/-, Mif-2-/-, and Cd74-/- mice had significantly worse tubular injury compared with wild-type (WT) control mice and that treatment with MIF-2/D-DT significantly improved recovery of injured epithelial cells. RNAseq analysis of kidney tissue from the ischemia-reperfusion injury model revealed that MIF-2/D-DT treatment stimulates secretory leukocyte proteinase inhibitor (SLPI) and cyclin D1 expression. MIF-2/D-DT additionally activates of eukaryotic initiation factor (eIF) 2α and activating transcription factor (ATF) 4, two transcription factors involved in the integrated stress response (ISR), which is a cellular stress response activated by hypoxia, nutrient deprivation, and oxygen radicals. MIF-2/D-DT also inhibited apoptosis and induced autophagy in hypoxia-treated mouse proximal tubular (MPT) cells. These results indicate that MIF-2/D-DT is an important factor in tubular cell regeneration and may be of therapeutic utility as a regenerative agent in the clinical setting of ischemic acute kidney injury. Copyright © 2017 the American Physiological Society.

  5. Diffeomorphic Multi-Frame Non-Rigid Registration of Cell Nuclei in 2D and 3D Live Cell Images.

    PubMed

    Tektonidis, Marco; Rohr, Karl

    2017-03-01

    To gain a better understanding of cellular and molecular processes, it is important to quantitatively analyze the motion of subcellular particles in live cell microscopy image sequences. Since, generally, the subcellular particles move and cell nuclei move as well as deform, it is important to decouple the movement of particles from that of the cell nuclei using non-rigid registration methods. We have developed a diffeomorphic multi-frame approach for non-rigid registration of cell nuclei in 2D and 3D live cell fluorescence microscopy images. Our non-rigid registration approach is based on local optic flow estimation, exploits information from multiple consecutive image frames, and determines diffeomorphic transformations in the log-domain, which allows efficient computation of the inverse transformations. To register single images of an image sequence to a reference image, we use a temporally weighted mean image, which is constructed based on inverse transformations and multiple consecutive frames. Using multiple consecutive frames improves the registration accuracy compared to pairwise registration, and using a temporally weighted mean image significantly reduces the computation time compared with previous work. In addition, we use a flow boundary preserving method for regularization of computed deformation vector fields, which prevents from over-smoothing compared to standard Gaussian filtering. Our approach has been successfully applied to 2D and 3D synthetic as well as real live cell microscopy image sequences, and an experimental comparison with non-rigid pairwise, multi-frame, and temporal groupwise registration has been carried out.

  6. Three dimensional spheroid cell culture for nanoparticle safety testing.

    PubMed

    Sambale, Franziska; Lavrentieva, Antonina; Stahl, Frank; Blume, Cornelia; Stiesch, Meike; Kasper, Cornelia; Bahnemann, Detlef; Scheper, Thomas

    2015-07-10

    Nanoparticles are widely employed for many applications and the number of consumer products, incorporating nanotechnology, is constantly increasing. A novel area of nanotechnology is the application in medical implants. The widespread use of nanoparticles leads to their higher prevalence in our environment. This, in turn, raises concerns regarding potential risks to humans. Previous studies have shown possible hazardous effects of some nanoparticles on mammalian cells grown in two-dimensional (2D) cultures. However, 2D in vitro cell cultures display several disadvantages such as changes in cell shape, cell function, cell responses and lack of cell-cell contacts. For this reason, the development of better models for mimicking in vivo conditions is essential. In the present work, we cultivated A549 cells and NIH-3T3 cells in three-dimensional (3D) spheroids and investigated the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO2-NP). The results were compared to cultivation in 2D monolayer culture. A549 cells in 3D cell culture formed loose aggregates which were more sensitive to the toxicity of ZnO-NP in comparison to cells grown in 2D monolayers. In contrast, NIH-3T3 cells showed a compact 3D spheroid structure and no differences in the sensitivity of the NIH-3T3 cells to ZnO-NP were observed between 2D and 3D cultures. TiO2-NP were non-toxic in 2D cultures but affected cell-cell interaction during 3D spheroid formation of A549 and NIH-3T3 cells. When TiO2-NP were directly added during spheroid formation in the cultures of the two cell lines tested, several smaller spheroids were formed instead of a single spheroid. This effect was not observed if the nanoparticles were added after spheroid formation. In this case, a slight decrease in cell viability was determined only for A549 3D spheroids. The obtained results demonstrate the importance of 3D cell culture studies for nanoparticle safety testing, since some effects cannot be revealed in 2D

  7. Mitotic arrest of breast cancer MDA-MB-231 cells by a halogenated thieno[3,2-d]pyrimidine.

    PubMed

    Ross, Christina R; Temburnikar, Kartik W; Wilson, Gerald M; Seley-Radtke, Katherine L

    2015-04-15

    Halogenated thieno[3,2-d]pyrimidines exhibit antiproliferative activity against a variety of cancer cell models, such as the mouse lymphocytic leukemia cell line L1210 in which they induce apoptosis independent of cell cycle arrest. Here we assessed these activities on MDA-MB-231 cells, a well-established model of aggressive, metastatic breast cancer. While 2,4-dichloro[3,2-d]pyrimidine was less toxic to MDA-MB-231 cells than previously observed in the L1210 model, flow cytometry analysis showed that MDA-MB-231 cell death involved arrest at the G2/M stage of the cell cycle. Conversely, the introduction of bromine at C7 of the 2,4-dichloro[3,2-d]pyrimidine eliminated cell type-dependent differences in cytotoxicity or cell cycle status. Together, these data indicate that a substituent at C7 can profoundly modify the cytotoxic mechanism of halogenated thieno[3,2-d]pyrimidines in a cell type-specific manner.

  8. Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling.

    PubMed

    Qin, Huilian; Lee, I-Fang; Panagiotopoulos, Constadina; Wang, Xiaoxia; Chu, Alvina D; Utz, Paul J; Priatel, John J; Tan, Rusung

    2011-03-01

    Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children. Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray. NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I-related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase-AKT pathway. These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.

  9. Expression of green fluorescent protein in human foreskin fibroblasts for use in 2D and 3D culture models.

    PubMed

    Chao, Jie; Peña, Tiffany; Heimann, Dean G; Hansen, Chris; Doyle, David A; Yanala, Ujwal R; Guenther, Timothy M; Carlson, Mark A

    2014-01-01

    The availability of fibroblasts that express green fluorescent protein (GFP) would be of interest for the monitoring of cell growth, migration, contraction, and other processes within the fibroblast-populated collagen matrix and other culture systems. A plasmid lentiviral vector-GFP (pLV-GFP) was utilized for gene delivery to produce primary human foreskin fibroblasts (HFFs) that stably express GFP. Cell morphology, cell migration, and collagen contraction were compared between nontransduced HFFs and transduced GFP-HFFs; no differences were observed. Immunocytochemical staining showed no differences in cell morphology between nontransduced and GFP-HFFs in both two-dimensional and three-dimensional culture systems. Furthermore, there was no significant difference in cellular population growth within the collagen matrix populated with nontransduced vs. GFP-HFFs. Within the limits of our assays, we conclude that transduction of GFP into HFFs did not alter the observed properties of HFFs compared with nontransduced fibroblasts. The GFP-HFFs may represent a new tool for the convenient monitoring of living primary fibroblast processes in two-dimensional or three-dimensional culture.

  10. Engineered bone culture in a perfusion bioreactor: a 2D computational study of stationary mass and momentum transport.

    PubMed

    Pierre, J; Oddou, C

    2007-12-01

    Successful bone cell culture in large implants still is a challenge to biologists and requires a strict control of the physicochemical and mechanical environments. This study analyses from the transport phenomena viewpoint the limiting factors of a perfusion bioreactor for bone cell culture within fibrous and porous large implants (2.5 cm in length, a few cubic centimetres in volume, 250 microm in fibre diameter with approximately 60% porosity). A two-dimensional mathematical model, based upon stationary mass and momentum transport in these implants is proposed and numerically solved. Cell oxygen consumption, in accordance theoretically with the Michaelis-Menten law, generates non linearity in the boundary conditions of the convection diffusion equation. Numerical solutions are obtained with a commercial code (Femlab 3.1; Comsol AB, Stockholm, Sweden). Moreover, based on the simplification of transport equations, a simple formula is given for estimating the length of the oxygen penetration within the implant. Results show that within a few hours of culture process and for a perfusion velocity of the order of 10(-4) m s(-1), the local oxygen concentration is everywhere sufficiently high to ensure a suitable cell metabolism. But shear stresses induced by the fluid flow with such a perfusion velocity are found to be locally too large (higher than 10(-3) Pa). Suitable shear stresses are obtained by decreasing the velocity at the inlet to around 2 x 10(-5) m s(-1). But consequently hypoxic regions (low oxygen concentrations) appear at the downstream part of the implant. Thus, it is suggested here that in the determination of the perfusion flow rate within a large implant, a compromise between oxygen supply and shear stress effects must be found in order to obtain a successful cell culture.

  11. Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials: an iTRAQ-coupled 2-D LC-MS/MS analysis.

    PubMed

    Xu, Jinling; Khor, Khiam Aik; Sui, Jianjun; Zhang, Jianhua; Tan, Tuan Lin; Chen, Wei Ning

    2008-10-01

    Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ-coupled 2-D LC-MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics.

  12. 2D Kinetic Particle in Cell Simulations of a Shear-Flow Stabilized Z-Pinch

    NASA Astrophysics Data System (ADS)

    Tummel, Kurt; Higginson, Drew; Schmidt, Andrea; Link, Anthony; McLean, Harry; Shumlak, Uri; Nelson, Brian; Golingo, Raymond; Claveau, Elliot; Lawrence Livermore National Lab Team; University of Washington Team

    2016-10-01

    The Z-pinch is a relatively simple and attractive potential fusion reactor design, but attempts to develop such a reactor have consistently struggled to overcome Z-pinch instabilities. The ``sausage'' and ``kink'' modes are among the most robust and prevalent Z-pinch instabilities, but theory and simulations suggest that axial flow-shear, dvz / dr ≠ 0 , can suppress these modes. Experiments have confirmed that Z-pinch plasmas with embedded axial flow-shear display a significantly enhanced resilience to the sausage and kink modes at a demonstration current of 50kAmps. A new experiment is under way to test the concept at higher current, and efforts to model these plasmas are being expanded. The performance and stability of these devices will depend on features like the plasma viscosity, anomalous resistivity, and finite Larmor radius effects, which are most accurately characterized in kinetic models. To predict these features, kinetic simulations using the particle in cell code LSP are now in development, and initial benchmarking and 2D stability analyses of the sausage mode are presented here. These results represent the first kinetic modeling of the flow-shear stabilized Z-pinch. This work is funded by the USDOE/ARPAe Alpha Program. Prepared by LLNL under Contract DE-AC52-07NA27344.

  13. 1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.

    PubMed

    Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N; Glenn, Sean T; Liu, Song; Trump, Donald L; Johnson, Candace S

    2015-04-01

    Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. miRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253J and 253J-BV cells express endogenous vitamin D receptor (VDR), which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  14. Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D

    PubMed Central

    Chaigne-Delalande, Benjamin; Li, Feng-Yen; O’Connor, Geraldine M.; Lukacs, Marshall J.; Jiang, Ping; Zheng, Lixin; Shatzer, Amber; Biancalana, Matthew; Pittaluga, Stefania; Matthews, Helen F.; Jancel, Timothy J.; Bleesing, Jack J.; Marsh, Rebecca A.; Kuijpers, Taco W.; Nichols, Kim E.; Lucas, Carrie L.; Nagpal, Sunil; Mehmet, Huseyin; Su, Helen C.; Cohen, Jeffrey I.; Uzel, Gulbu; Lenardo, Michael J.

    2013-01-01

    The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free Mg2+ levels. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg2+ causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8+ T cells and impairs cytolytic responses against EBV. Remarkably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg2+ and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg2+ in eukaryotic cells. PMID:23846901

  15. Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D.

    PubMed

    Chaigne-Delalande, Benjamin; Li, Feng-Yen; O'Connor, Geraldine M; Lukacs, Marshall J; Jiang, Ping; Zheng, Lixin; Shatzer, Amber; Biancalana, Matthew; Pittaluga, Stefania; Matthews, Helen F; Jancel, Timothy J; Bleesing, Jack J; Marsh, Rebecca A; Kuijpers, Taco W; Nichols, Kim E; Lucas, Carrie L; Nagpal, Sunil; Mehmet, Huseyin; Su, Helen C; Cohen, Jeffrey I; Uzel, Gulbu; Lenardo, Michael J

    2013-07-12

    The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg(2+)) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg(2+) causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8(+) T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg(2+) and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg(2+) in eukaryotic cells.

  16. FDTD analysis of 2D triangular-lattice photonic crystals with arbitrary-shape inclusions based on unit cell transformation

    NASA Astrophysics Data System (ADS)

    Ma, Zetao; Ogusu, Kazuhiko

    2009-04-01

    A finite-difference time-domain method based on Yee's orthogonal cell is utilized to calculate the band structures of 2D triangular-lattice-based photonic crystals through a simple modification to properly shifting the boundaries of the original unit cell. A strategy is proposed for transforming the triangular unit cell into an orthogonal one, which can be used to calculate the band structures of 2D PhCs with various shapes of inclusions, such as triangular, quadrangular, and hexagonal shapes, to overcome the shortage of plane-wave expansion method for circular one. The band structures of 2D triangular-lattice-based PhCs with hexagonal air-holes are calculated and discussed for different values of its radius and rotation angle. The obtained results provide an insight to manipulate the band structures of PhCs.

  17. Perfusion Based Cell Culture Chips

    NASA Astrophysics Data System (ADS)

    Heiskanen, A.; Emnéus, J.; Dufva, M.

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.

  18. 1-integrin and MT1-MMP promote tumor cell migration in 2D but not in 3D fibronectin microenvironments

    NASA Astrophysics Data System (ADS)

    Corall, Silke; Haraszti, Tamas; Bartoschik, Tanja; Spatz, Joachim Pius; Ludwig, Thomas; Cavalcanti-Adam, Elisabetta Ada

    2014-03-01

    Cell migration is a crucial event for physiological processes, such as embryonic development and wound healing, as well as for pathological processes, such as cancer dissemination and metastasis formation. Cancer cell migration is a result of the concerted action of matrix metalloproteinases (MMPs), expressed by cancer cells to degrade the surrounding matrix, and integrins, the transmembrane receptors responsible for cell binding to matrix proteins. While it is known that cell-microenvironment interactions are essential for migration, the role of the physical state of such interactions remains still unclear. In this study we investigated human fibrosarcoma cell migration in two-dimensional (2D) and three-dimensional (3D) fibronectin (FN) microenvironments. By using antibody blocking approach and cell-binding site mutation, we determined that -integrin is the main mediator of fibrosarcoma cell migration in 2D FN, whereas in 3D fibrillar FN, the binding of - and -integrins is not necessary for cell movement in the fibrillar network. Furthermore, while the general inhibition of MMPs with GM6001 has no effect on cell migration in both 2D and 3D FN matrices, we observed opposing effect after targeted silencing of a membrane-bound MMP, namely MT1-MMP. In 2D fibronectin, silencing of MT1-MMP results in decreased migration speed and loss of directionality, whereas in 3D FN matrices, cell migration speed is increased and integrin-mediated signaling for actin dynamics is promoted. Our results suggest that the fibrillar nature of the matrix governs the migratory behavior of fibrosarcoma cells. Therefore, to hinder migration and dissemination of diseased cells, matrix molecules should be directly targeted, rather than specific subtypes of receptors at the cell membrane.

  19. Crosstalk between SOXB1 proteins and WNT/β-catenin signaling in NT2/D1 cells.

    PubMed

    Mojsin, Marija; Topalovic, Vladanka; Vicentic, Jelena Marjanovic; Schwirtlich, Marija; Stanisavljevic, Danijela; Drakulic, Danijela; Stevanovic, Milena

    2015-11-01

    During early vertebrate embryogenesis, the expression of SOXB1 proteins is precisely regulated by a number of different mechanisms, including Wnt/β-catenin signaling. This is essential for controlling the balance between stemness and differentiation in embryonic stem cells. In the present study, we analyzed the molecular mechanism of LiCl action in NT2/D1 cells and examined the crosstalk between SOXB1 proteins and Wnt signaling in this model system. We have shown that LiCl increases β-catenin level, induces its translocation to the nucleus and consequently up-regulates β-catenin/Tcf-dependent transcription in NT2/D1 cells. Our results also suggest that LiCl treatment leads to increased expression of SOX2 and SOX3 proteins in NT2/D1 cells through activation of canonical Wnt signaling. Finally, we have detected a negative feedback loop between β-catenin and SOX2 expression in NT2/D1 cells. Since β-catenin and SOX2 have been linked to processes of self-renewal and pluripotency, our results have implications for future research on the maintenance of stemness and lineage commitment of embryonic stem cells.

  20. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  1. Theoretical studies of effects of 2D plasmonic grating on electrical properties of organic solar cells

    NASA Astrophysics Data System (ADS)

    Sha, Wei E. I.; Choy, Wallace C. H.; Chew, Weng Cho

    2012-09-01

    Although various optical designs and physical mechanisms have been studied both experimentally and theoretically to improve the optical absorption of organic solar cells (OSCs) by incorporating metallic nanostructures, the effects of plasmonic nanostructures on the electrical properties of OSCs is still not fully understood. Hence, it is highly desirable to study the changes of electrical properties induced by plasmonic structures and the corresponding physics for OSCs. In this work, we develop a multiphysics model for plasmonic OSCs by solving the Maxwell's equations and semiconductor equations (Poisson, continuity, and drift-diffusion equations) with unified finite-difference method. Both the optical and electrical properties of OSCs incorporating a 2D metallic grating anode are investigated. For typical active polymer materials, low hole mobility, which is about one magnitude smaller than electron mobility, dominates the electrical property of OSCs. Since surface plasmon resonances excited by the metallic grating will produce concentrated near-field penetrated into the active polymer layer and decayed exponentially away from the metal-polymer interface, a significantly nonuniform and extremely high exciton generation rate is obtained near the grating. Interestingly, the reduced recombination loss and the increased open-circuit voltage can be achieved in plasmonic OSCs. The physical origin of the phenomena lies at direct hole collections to the metallic grating anode with a short transport path. In comparison with the plasmonic OSC, the hole transport in a multilayer planar OSC experiences a long transport path and time because the standard planar OSC has a high exciton generation rate at the transparent front cathode. The unveiled multiphysics is particularly helpful for designing high-performance plasmonic OSCs.

  2. NEDD9 stabilizes focal adhesions, increases binding to the extra-cellular matrix and differentially effects 2D versus 3D cell migration.

    PubMed

    Zhong, Jessie; Baquiran, Jaime B; Bonakdar, Navid; Lees, Justin; Ching, Yu Wooi; Pugacheva, Elena; Fabry, Ben; O'Neill, Geraldine M

    2012-01-01

    The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 -/- MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9-/- MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9-/- MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9-/-, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.

  3. The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

    PubMed

    Drakulic, Danijela; Vicentic, Jelena Marjanovic; Schwirtlich, Marija; Tosic, Jelena; Krstic, Aleksandar; Klajn, Andrijana; Stevanovic, Milena

    2015-03-01

    The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

  4. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    PubMed

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  5. Notch- and vitamin D signaling in 1,25(OH)2D3-resistant glioblastoma multiforme (GBM) cell lines.

    PubMed

    Reichrath, Sandra; Müller, Cornelia S L; Gleissner, Beate; Pfreundschuh, Michael; Vogt, Thomas; Reichrath, Jörg

    2010-07-01

    Recently, an important role of Notch activation for Ras-induced transformation of glial cells and for glioma growth and survival has been demonstrated. It was concluded that activation of Notch-signaling may represent a new target for glioblastoma multiforme (GBM) therapy. We now analyzed five GBM cell lines (Tx3095, Tx3868, U87, U118, U373) for key components of Notch-signaling pathways (Notch-1, Notch-2, Notch-3, Notch-4, Delta-like 1, Delta-like 3, Delta-like 4, Jagged-1, Jagged-2) using conventional RT-PCR. We found that some components (Notch-1, Notch-2, Notch-4, Jagged-1) were consistently expressed in all cell lines analyzed while, in contrast, other key components of Notch-signaling were differentially expressed. Notch-3 was expressed in three out of five cell lines (in U87, U118 and U373), but was missing in Tx3095 and Tx3868 cells. Jagged-2 was expressed in U87, U373 and Tx3868, but not in U118 or Tx3095 cells. Delta-like 1 and Delta-like 3 were not detected in Tx3905 cells, but in all other cell lines. RNA for Delta-like 4 was only found in U373 and Tx3868 GBM cell lines. Treating GBM cell lines with 1,25(OH)2D3 (10(-6), 10(-8), and 10(-10) M), the biologically active form of vitamin D, did not result in significant dose- or time-dependent antiproliferative effects, indicating that GBM cell lines are resistant against the antiproliferative activity of 1,25(OH)2D3. In vitro treatment of GBM cells with 1,25(OH)2D3 did not result in a modulation of the expression of key components of the Notch-signaling pathway. Treatment with HDAC-inhibitor TSA or DNA-methyltransferase inhibitor 5-aza exerted dose- and time-dependent antiproliferative effects on GBM cell lines. We asked the question whether the resistance against 1,25(OH)2D3 could be restored by co-treatment with TSA or 5-aza. However, combination therapy with 1,25(OH)2D3 and TSA or 5-aza did not result in enhanced antiproliferative effects as compared to treatment with TSA or 5-aza alone. In contrast

  6. NKG2D/Ligand dysregulation and functional alteration of innate immunity cell populations in pediatric IBD.

    PubMed

    La Scaleia, Raffaella; Stoppacciaro, Antonella; Oliva, Salvatore; Morrone, Stefania; Di Nardo, Giovanni; Santoni, Angela; Cucchiara, Salvatore; Palmieri, Gabriella

    2012-10-01

    Dysregulated innate immune responses play an important role in inflammatory bowel disease (IBD). NKG2D innate immunity receptor is a major sensor of tissue damage that, by recognizing multiple stress-induced, cell-associated ligands (MIC-A/B and ULBP1-5), potentiates the effector functions of "innate-like" (γ/δ TcR+, and natural killer receptor+ [NKR+]) T-cell populations. We analyzed the representivity, NKG2D/ligand expression pattern, and functional ability of the major innate immunity cell populations in pediatric IBD patients. We analyzed 41 Crohn's disease (CD) patients, 33 ulcerative colitis (UC) patients, and 51 age-matched non-IBD controls. The expression of NKG2D and its ligands, interferon-gamma (IFN-γ) production, and cytotoxic granule release were assessed by immunostaining and multiparameter cytofluorimetric analysis on circulating and mucosal mononuclear subsets; the inflammatory infiltrate was also characterized by immunohistochemistry. The expression pattern of NKG2D receptor and its ligands on mucosal and circulating innate immunity populations is severely disturbed in IBD; NKG2D and ligands are upregulated on immune infiltrate in both CD and UC active lesions; receptor/ligand upregulation also occurs on circulating leukocyte populations, where it depends on both disease activity and type (UC vs. CD). Finally, the frequency and effector capability of peripheral blood "innate-like" T-cell populations are also altered in IBD patients. The circulating and mucosal innate immunity compartment is phenotypically and functionally altered in pediatric IBD; some alterations may represent a distinctive feature of the pediatric disease condition. The disturbance of NKG2D/ligand pathway may play a role in sustaining immune activation which leads to chronic inflammatory tissue damage. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

  7. Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

    PubMed

    Chang, Chih-Jung; Chen, Yi-Yuan M; Lu, Chia-Chen; Lin, Chuan-Sheng; Martel, Jan; Tsai, Sheng-Hui; Ko, Yun-Fei; Huang, Tsung-Teng; Ojcius, David M; Young, John D; Lai, Hsin-Chih

    2014-04-01

    Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

  8. Differential gene expression by 1,25(OH)2D3 in an endometriosis stromal cell line.

    PubMed

    Ingles, Sue Ann; Wu, Liang; Liu, Benjamin T; Chen, Yibu; Wang, Chun-Yeh; Templeman, Claire; Brueggmann, Doerthe

    2017-01-28

    Endometriosis is a common female reproductive disease characterized by invasion of endometrial cells into other organs, frequently causing pelvic pain and infertility. Alterations of the vitamin D system have been linked to endometriosis incidence and severity. To shed light on the potential mechanism for these associations, we examined the effects of 1,25(OH)2D3 on gene expression in endometriosis cells. Stromal cell lines derived from endometriosis tissue were treated with 1,25(OH)2D3, and RNA-seq was used to identify genes differentially expressed between treated and untreated cells. Gene ontology and pathway analyses were carried out using Partek Flow and Ingenuity software suites, respectively. We identified 1627 genes that were differentially expressed (886 down-regulated and 741 up-regulated) by 1,25(OH)2D3. Only one gene, CYP24A1, was strongly up-regulated (369-fold). Many genes were strongly down-regulated. 1,25(OH)2D3 treatment down-regulated several genetic pathways related to neuroangiogenesis, cellular motility, and invasion, including pathways for axonal guidance, Rho GDP signaling, and matrix metalloprotease inhibition. These findings support a role for vitamin D in the pathophysiology of endometriosis, and provide new targets for investigation into possible causes and treatments.

  9. PPARγ suppresses the proliferation of cardiac myxoma cells through downregulation of MEF2D in a miR-122-dependent manner

    SciTech Connect

    Qiu, Youzhu; Yang, Jie; Bian, Shizhu; Chen, Guozhu; Yu, Jie

    2016-06-03

    Peroxisome proliferator-activated receptor gamma (PPARγ), a multiple functional transcription factor, has been reported to have anti-tumor effects through inhibition of cells proliferation. However, its effects on cardiac myxoma (CM) cells and the underlying signaling mechanism is unclear. In the present study, we demonstrated that the level of PPARγ is inversely correlated with that of myocyte enhancer factor 2D (MEF2D), a biomarker of CM. We found that activation of PPARγ inhibit MEF2D expression via upregulation of miR-122, which can target the 3′-UTR of MEF2D and inhibit MEF2D expression, by directly binding to the PPRE in the miR-122 promoter region. Functional experiments further showed that miR-122-dependent downregulation of MEF2D by PPARγ suppress the proliferation of CM cells. These results suggest that PPARγ may exert its antiproliferative effects by negatively regulating the MEF2D in CM cells, which through upregulation of miR-122, and PPARγ/miR-122/MEF2D signaling pathway may be a novel target for treatment of CM. -- Highlights: •PPARγ expression is inversely correlated with MEF2D expression in CM tissues. •PPARγ downregulates MEF2D expression in CM cells. •PPARγ inhibits MEF2D expression via upregulation of miR-122. •miR-122-dependent downregulation of MEF2D by PPARγ suppresses the proliferation of CM cells.

  10. NKG2D stimulation of CD8+ T cells during priming promotes their capacity to produce cytokines in response to viral infection in mice.

    PubMed

    Kavazović, Inga; Lenartić, Maja; Jelenčić, Vedrana; Jurković, Slaven; Lemmermann, Niels A W; Jonjić, Stipan; Polić, Bojan; Wensveen, Felix M

    2017-04-04

    NKG2D is an activating receptor that is expressed on most cytotoxic cells of the immune system, including NK cells, γδ and CD8(+) T cells. It is still a matter of debate whether and how NKG2D mediates priming of CD8(+) T cells in vivo, due to a lack of studies where NKG2D is eliminated exclusively in these cells. Here we studied the impact of NKG2D on effector CD8(+) T-cell formation. NKG2D-deficiency that is restricted to murine CD8(+) T cells did not impair antigen-specific T-cell expansion following mCMV and LCMV infection, but reduced their capacity to produce cytokines. Upon infection, conventional dendritic cells induce NKG2D ligands, which drive cytokine production on CD8(+) T cells via the Dap10 signaling pathway. T-cell development, homing and proliferation were not affected by NKG2D deficiency and cytotoxicity was only impaired when strong T-cell receptor stimuli were used. Transfer of antigen-specific CD8(+) T cells demonstrated that NKG2D-deficiency attenuated their capacity to reduce viral loads. The inability of NKG2D-deficient cells to produce cytokines could be overcome with injection of IL-15 super-agonist during priming. In summary, our data shows that NKG2D has a non-redundant role in priming of CD8(+) T cells to produce antiviral cytokines. Upon viral infection, classical Dendritic cells induce expression of the NKG2D ligand H60. NKG2D stimulation during priming enhances the ability of CD8 T cells to produce cytokines but not increases cytotoxic potential upon T cell receptor engagement in the periphery. This article is protected by copyright. All rights reserved.

  11. A Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Protocol for Studies of Neural Precursor Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of up to distinct proteomes. The example given describes the analysis of undifferentiated and differentiated neural precursor cells labelled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labelled with Cy2. After labelling, the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay of the two different proteomes and the differences in abundance of specific protein spots can be determined through comparison to the pooled standard.

  12. Search for limiting factors in the RNAi pathway in silkmoth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and Translin.

    PubMed

    Swevers, Luc; Liu, Jisheng; Huvenne, Hanneke; Smagghe, Guy

    2011-01-01

    RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

  13. 2D/3D perovskite hybrids as moisture-tolerant and efficient light absorbers for solar cells.

    PubMed

    Ma, Chaoyan; Leng, Chongqian; Ji, Yixiong; Wei, Xingzhan; Sun, Kuan; Tang, Linlong; Yang, Jun; Luo, Wei; Li, Chaolong; Deng, Yunsheng; Feng, Shuanglong; Shen, Jun; Lu, Shirong; Du, Chunlei; Shi, Haofei

    2016-11-03

    The lifetime and power conversion efficiency are the key issues for the commercialization of perovskite solar cells (PSCs). In this paper, the development of 2D/3D perovskite hybrids (CA2PbI4/MAPbIxCl3-x) was firstly demonstrated to be a reliable method to combine their advantages, and provided a new concept for achieving both stable and efficient PSCs through the hybridization of perovskites. 2D/3D perovskite hybrids afforded significantly-improved moisture stability of films and devices without encapsulation in a high humidity of 63 ± 5%, as compared with the 3D perovskite (MAPbIxCl3-x). The 2D/3D perovskite-hybrid film did not undergo any degradation after 40 days, while the 3D perovskite decomposed completely under the same conditions after 8 days. The 2D/3D perovskite-hybrid device maintained 54% of the original efficiency after 220 hours, whereas the 3D perovskite device lost all the efficiency within only 50 hours. Moreover, the 2D/3D perovskite hybrid achieved comparable device performances (PCE: 13.86%) to the 3D perovskite (PCE: 13.12%) after the optimization of device fabrication conditions.

  14. Three-dimensional cell culturing by magnetic levitation.

    PubMed

    Haisler, William L; Timm, David M; Gage, Jacob A; Tseng, Hubert; Killian, T C; Souza, Glauco R

    2013-10-01

    Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

  15. An MEK-cofilin signalling module controls migration of human T cells in 3D but not 2D environments.

    PubMed

    Klemke, Martin; Kramer, Elisabeth; Konstandin, Mathias H; Wabnitz, Guido H; Samstag, Yvonne

    2010-09-01

    T cells infiltrate peripheral tissues to execute immunosurveillance and effector functions. For this purpose, T cells first migrate on the two-dimensional (2D) surface of endothelial cells to undergo transendothelial migration. Then they change their mode of movement to undergo migration within the three-dimensional (3D)-extracellular matrix of the infiltrated tissue. As yet, no molecular mechanisms are known, which control migration exclusively in either 2D or 3D environments. Here, we describe a signalling module that controls T-cell chemotaxis specifically in 3D environments. In chemotaxing T cells, Ras activity is spatially restricted to the lamellipodium. There, Ras initiates activation of MEK, which in turn inhibits LIM-kinase 1 activity, thereby allowing dephosphorylation of the F-actin-remodelling protein cofilin. Interference with this MEK-cofilin module by either inhibition of MEK or by knockdown of cofilin reduces speed and directionality of chemotactic migration in 3D-extracellular matrices, but not on 2D substrates. This MEK-cofilin module may have an important function in the tissue positioning of T cells during an immune response.

  16. Proteomic analysis of estrogen response of premalignant human breast cells using a 2-D liquid separation/mass mapping technique.

    PubMed

    Zhao, Jia; Zhu, Kan; Lubman, David M; Miller, Fred R; Shekhar, Malthy P V; Gerard, Brigitte; Barder, Timothy J

    2006-07-01

    A 2-D liquid-phase separation method based on chromatofocusing and nonporous silica RP-HPLC followed by ESI-TOF-MS was used to analyze proteins in whole cell lysates from estrogen-treated and untreated premalignant, estrogen-responsive cell line MCF10AT1 cells. 2-D mass maps in the pH range 4.6-6.0 were generated with good correlation to theoretical M(r) values for intact proteins. Proteins were identified based on intact M(r), pI and PMF, or MS/MS sequencing. About 300 unique proteins were identified and 120 proteins in mass range 5-75 kDa were quantified upon treatment of estrogen. Around 40 proteins were found to be more highly expressed (>four-fold) and 17 were down-regulated (>four-fold) in treated cells. In our study, we found that many altered proteins have characteristics consistent with the development of a malignant phenotype. Some of them have a role in the ras pathway or play an important role in signal pathways. These changed proteins might be essential in the estrogen regulation mechanism. Our study highlights the use of the MCF10AT1 cell line to examine estrogen-induced changes in premalignant breast cells and the ability of the 2-D mass mapping technique to quantitatively study protein expression changes on a proteomic scale.

  17. Influence of basement membrane proteins and endothelial cell-derived factors on the morphology of human fetal-derived astrocytes in 2D.

    PubMed

    Levy, Amanda F; Zayats, Maya; Guerrero-Cazares, Hugo; Quiñones-Hinojosa, Alfredo; Searson, Peter C

    2014-01-01

    Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB), however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF), laminin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF). Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF) to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models.

  18. Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7

    PubMed Central

    Granum, Stine; Sundvold-Gjerstad, Vibeke; Dai, Ke-Zheng; Kolltveit, Kristin Melkevik; Hildebrand, Kjersti; Huitfeldt, Henrik S; Lea, Tor; Spurkland, Anne

    2006-01-01

    Background The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. Results TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. Conclusion These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined. PMID:16839418

  19. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk.

  20. Mammalian Cell Tissue Culture Techniques.

    PubMed

    Phelan, Katy; May, Kristin M

    2016-06-01

    Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  1. Activation of vitamin D receptor (VDR)- and peroxisome proliferator-activated receptor (PPAR)-signaling pathways through 1,25(OH)(2)D(3) in melanoma cell lines and other skin-derived cell lines.

    PubMed

    Sertznig, Pit; Seifert, Markus; Tilgen, Wolfgang; Reichrath, Jörg

    2009-07-01

    We have investigated expression of vitamin D receptor (VDR) and peroxisome proliferator-activated receptors (PPAR)alpha, delta, gamma in primary cultured normal melanocytes (NHM), melanoma cell lines (MeWo, SK-Mel-5, SK-Mel-25, SK-Mel-28), a cutaneous squamous cell carcinoma cell line (SCL-1) and an immortalized sebocyte cell line (SZ95). LNCaP prostate cancer cells, MCF-7 breast cancer cells and embryonic kidney cells (HEK-293) were used as controls. VDR and PPAR mRNA were detected, quantitated and compared in these cell lines using real-time quantitative polymerase chain reaction (RTqPCR). The expression patterns of these nuclear receptors (NRs) varied strongly between the different cell lines according to their origin. PPARdelta and PPARgamma were less strongly expressed in the melanoma cell lines and in the other skin-derived cell lines as compared to the control cell lines. PPARalpha and VDR were stronger expressed in the 1,25(OH)(2)D(3)-sensitive melanoma cells (MeWo and in SK-Mel-28) than in the 1,25(OH)(2)D(3)-resistent melanoma cell lines (SK-Mel-5 and SK-Mel-25) or in NHM. Interestingly, VDR expression was increased by the treatment with 1,25(OH)(2)D(3) in 1,25(OH)(2)D(3)-sensitive melanoma cells but not in 1,25(OH)(2)D(3)-resistent melanoma cell lines. 1,25(OH)(2)D(3) increased the expression of PPARalpha in almost all cell lines analyzed. Our results indicate a cross-talk between VDR- and PPAR-signaling pathways in various cell types including melanoma cells. Further investigations are required to investigate the physiological and pathophysiological relevance of this cross-talk. Because VDRand PPAR-signaling pathways regulate a multitude of genes that are of importance for a multitude of cellular functions including cell proliferation, cell differentiation, immune responses and apoptosis, the provided link between VDR and PPAR may open important new perspectives for treatment and prevention of melanoma and other diseases.

  2. STAT3 contributes to NK cell recognition by modulating expression of NKG2D ligands in adriamycin-resistant K562/AO2 cells.

    PubMed

    Cai, Xiaohui; Lu, Xuzhang; Jia, Zhuxia; Zhang, Xiuwen; Han, Wenmin; Rong, Xiao; Ma, Lingdi; Zhou, Min; Chen, Baoan

    2015-11-01

    Leukemic cells can survive after chemotherapy by acquisition of multidrug resistance genes, but other phenotypes related to escape from immune recognition remain elusive. Adriamycin-resistant K562/AO2 cells are less susceptible to elimination by NK cells compared with wild type K562 cells due to lower expression of NKG2D ligands. Treatment of K562/AO2 cells with STAT3 inhibitor VII resulted in reduced expression of multidrug resistance gene P-glycoprotein, and up-regulation of NKG2D ligands on K562/AO2 cells. Meanwhile, K562/AO2 cells treated with STAT3 inhibitor proliferated less and were more susceptible to killing by NK cells than untreated K562/AO2 cells. The enhanced cytotoxicity of NK cells against K562/AO2 cells was partly blocked by treatment of NK cells with anti-NKG2D antibodies. These data suggest that STAT3 contributes to NK cell recognition by modulating NKG2D ligands in K562/AO2 cells, which may a mechanism by which cells survive and cause relapse of leukemia.

  3. Vinculin Regulates Directionality and Cell Polarity in 2D, 3D Matrix and 3D Microtrack Migration.

    PubMed

    Rahman, Aniqua; Carey, Shawn P; Kraning-Rush, Casey M; Goldblatt, Zachary E; Bordeleau, Francois; Lampi, Marsha C; Lin, Deanna Y; García, Andrés J; Reinhart-King, Cynthia A

    2016-03-09

    During metastasis, cells can use proteolytic activity to form tube-like "microtracks" within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro 3D micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Since focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on 2D substrates and in 3D uniform collagen matrices, indicated by reduced speed, shorter net displacement and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for Focal Adhesion Kinase (FAK) activation in 3D as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks, but not on 2D substrates, and accordingly, FAK inhibition halts cell migration in 3D microtracks. Together, these data indicate that vinculin plays a key role in polarization during migration.

  4. Three-dimensional Tissue Culture Based on Magnetic Cell Levitation

    PubMed Central

    Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.

    2015-01-01

    Cell culture is an essential tool for drug discovery, tissue engineering, and stem cell research. Conventional tissue culture produces two-dimensional (2D) cell growth with gene expression, signaling, and morphology that can differ from those in vivo and thus compromise clinical relevancy1–5. Here we report a three-dimensional (3D) culture of cells based on magnetic levitation in the presence of hydrogels containing gold and magnetic iron oxide (MIO) nanoparticles plus filamentous bacteriophage. This methodology allows for control of cell mass geometry and guided, multicellular clustering of different cell types in co-culture through spatial variance of the magnetic field. Moreover, magnetic levitation of human glioblastoma cells demonstrates similar protein expression profiles to those observed in human tumor xenografts. Taken together, these results suggest levitated 3D culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and allows for long-term multi-cellular studies. PMID:20228788

  5. Preservation of the 3D Phenotype Upon Dispersal of Cultured Cell Spheroids Into Monolayer Cultures.

    PubMed

    Koshkin, Vasilij; Ailles, Laurie E; Liu, Geoffrey; Krylov, Sergey N

    2017-01-01

    In functional cytometric studies, cultured cells are exposed to effectors (e.g., drugs), and the heterogeneity of cell responses are studied using cytometry techniques (e.g., image cytometry). Such studies are difficult to perform on 3D cell cultures. A solution is to disperse 3D clusters and transfer the cells to the 2D state before applying effectors and using cytometry. This approach requires that the lifetime of the 3D phenotype be longer than the duration of the experiment. Here we studied the dynamics of phenotype transformation from 3D to 2D and searched for means of slowing this transformation down in dispersed spheroids of MCF7 cells. We found three functional biomarkers of the 3D phenotype in MCF7 cell spheroids that are absent in the 2D cell culture: (i) the presence of a subpopulation with an elevated drug-expelling capacity; (ii) the presence of a subpopulation with an elevated cytoprotective capacity; and (iii) the accumulation of cells in the G1 phase of the cell cycle. Monitoring these biomarkers in cells transferred from the 3D state to the 2D state revealed their gradual extinction. We found that the combined application of an elevated cell density and thiol-containing medium supplements increased the lifetime of the 3D phenotype by several fold to as long as 96 h. Our results suggest that extending the lifetime of the 3D phenotype in the cells transferred from the 3D state to the 2D state can facilitate detailed functional cytometric studies, such as measurements of population heterogeneity of cytotoxicity, chemosensitivity, and radiosensitivity. J. Cell. Biochem. 118: 154-162, 2017. © 2016 Wiley Periodicals, Inc.

  6. 2D protrusion but not motility predicts growth factor-induced cancer cell migration in 3D collagen.

    PubMed

    Meyer, Aaron S; Hughes-Alford, Shannon K; Kay, Jennifer E; Castillo, Amalchi; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A

    2012-06-11

    Growth factor-induced migration is a critical step in the dissemination and metastasis of solid tumors. Although differences in properties characterizing cell migration on two-dimensional (2D) substrata versus within three-dimensional (3D) matrices have been noted for particular growth factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially for high-throughput experiments. We therefore were motivated to investigate which migration properties measured in various 2D assays might be reflective of 3D migratory behavioral responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration properties did not correlate well with 3D behavior across multiple growth factors, we found that increased membrane protrusion elicited by growth factor stimulation did relate robustly to enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we observed this to be a more reliable relationship than cognate receptor expression or activation levels across these and two additional mammary tumor lines.

  7. Enhanced photovoltaic properties of dye-sensitized solar cell based on ultrathin 2D TiO2 nanostructures

    NASA Astrophysics Data System (ADS)

    Zhang, Putao; Hu, Zhiqiang; Wang, Yan; Qin, Yiying; Sun, Xiao Wei; Li, Wenqin; Wang, Jinmin

    2016-04-01

    Ultrathin two-dimensional (2D) TiO2 nanostructures with a thickness of ∼5 nm and a specific surface area of 257.3 m2 g-1 were synthesized by a hydrothermal process. The 2D TiO2 nanostructures and P25 nanoparticles were introduced as scattering layer and underlayer to construct a bi-layer photoanode in a dye-sensitized solar cell (DSSC). The as-prepared DSSC exhibits an enhanced power conversion efficiency (5.14%), which is 23.9% higher than that of pure P25 DSSC (4.15%). Electrochemical impedance spectroscopy (EIS) indicates that DSSC based on P25-2D TiO2 nanostructures shows a longer life time and a larger recombination resistance. The enhanced photovoltaic properties are attributed to the excellent light scattering capability and high capacity for dye adsorption of 2D TiO2 nanostructures, which makes them a promising candidate as an efficient scattering layer in high-performance DSSCs.

  8. Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

    PubMed Central

    Proff, Julia; Schaft, Niels; Dörrie, Jan; Full, Florian; Ensser, Armin; Muller, Yves A.; Cerwenka, Adelheid; Abken, Hinrich; Parolini, Ornella; Ambros, Peter F.; Kovar, Heinrich; Holter, Wolfgang

    2012-01-01

    We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection. PMID:22355347

  9. Scorpion venom activates natural killer cells in hepatocellular carcinoma via the NKG2D-MICA pathway.

    PubMed

    Chen, Han; Zhidan, Wang; Xia, Ren; Zhaoxia, Wang; Qing, Jia; Qiang, Guo; Haipeng, Yin; Hengxiao, Wang

    2016-06-01

    Previous studies have demonstrated that polypeptides extracted from scorpion venom (PESV) inhibited cell proliferation in several tumors, however, the effect on dysfunctional and exhausted natural killer cells which contribute to tumor escape from immune surveillance remain to be elucidated. In this study, we determined the effect of PESV on NK infiltration into H22 cells orthotopic transplantation tumors and on the expression of MHC class I chain-related proteins A (MICA) in HepG2 cells. We found that tumor growth in mice was significantly inhibited by PESV and the survival time of tumor-bearing mice treated with PESV was significantly prolonged. Moreover, levels of tumor-infiltrating NK cells, NKG2D protein, perforin and granzyme B mRNA were significantly increased in the group treated with PESV compared with the tumor-bearing control group. In addition, In addition, up-regulation of MICA by PESV enhances the susceptibility of HepG2 cells to NK lysis in vitro. These results indicate that the inhibitory effects of PESV on hepatic carcinoma are likely mediated by up-regulation of NK cell activity via the MICA-NKG2D pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. The histone deacetylase inhibitor valproic acid inhibits NKG2D expression in natural killer cells through suppression of STAT3 and HDAC3

    PubMed Central

    Ni, Lulu; Wang, Lixin; Yao, Chao; Ni, Zhongya; Liu, Fei; Gong, Chenyuan; Zhu, Xiaowen; Yan, Xuewei; Watowich, Stephanie S.; Lee, Dean A.; Zhu, Shiguo

    2017-01-01

    NKG2D is a major activating receptor of NK cells and plays a critical role in tumor immunosurveillance. NKG2D expression in NK cells is inhibited by the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and enhanced by the narrow-spectrum HDAC inhibitor entinostat. We previously demonstrated that entinostat enhanced NKG2D transcription by increasing acetylation of Histones H3 and H4. However, the mechanism by which VPA reduces NKG2D expression in NK cells is not known. We have also shown that NKG2D transcription is regulated by STAT3 phosphorylation. In this study, we investigated regulation of NKG2D expression in NK cells by VPA and entinostat by assessing protein expression, phosphorylation, and interaction of HDACs and STAT3. We find that VPA selectively inhibits STAT3 tyrosine705 phosphorylation, but entinostat does not. STAT3 complexes with HDAC3, and HDAC3 inhibition represses STAT3 phosphorylation and therefore NKG2D expression. NK cells from STAT3 wild-type mice downregulate NKG2D in response to VPA, but not NK cells from STAT3 knockout mice. These results show that VPA is a potent inhibitor of STAT3 phosphorylation and demonstrate that histone acetylation and STAT3 tyrosine705 phosphorylation cooperate in regulating NKG2D expression in NK cells. PMID:28338101

  11. Genotoxic Stress Induces Senescence-Associated ADAM10-Dependent Release of NKG2D MIC Ligands in Multiple Myeloma Cells.

    PubMed

    Zingoni, Alessandra; Cecere, Francesca; Vulpis, Elisabetta; Fionda, Cinzia; Molfetta, Rosa; Soriani, Alessandra; Petrucci, Maria Teresa; Ricciardi, Maria Rosaria; Fuerst, Daniel; Amendola, Maria Giulia; Mytilineos, Joannis; Cerboni, Cristina; Paolini, Rossella; Cippitelli, Marco; Santoni, Angela

    2015-07-15

    Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor recognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I-related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM) cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is upregulated upon chemotherapeutic treatment both in patient-derived CD138(+)/CD38(+) plasma cells and in several MM cell lines, and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs and metalloproteinase inhibitors enhances NK cell-mediated recognition of MM cells, preserving MIC molecules on the cell surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell-based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune responses. Copyright © 2015 by The American Association of Immunologists, Inc.

  12. Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12.

    PubMed

    Girart, María V; Fuertes, Mercedes B; Domaica, Carolina I; Rossi, Lucas E; Zwirner, Norberto W

    2007-09-15

    NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.

  13. Interpretation of cell culture phenomena.

    PubMed

    Vierck, J L; Dodson, M V

    2000-03-01

    This paper discusses the dilemma of interpreting unusual or abnormal phenomena seen in cell cultures and is not intended to address the statistical design of experiments. Problems that can be encountered when growing cells in experimental situations include low or decreasing cell numbers, abnormal cell morphology, microbial contamination, and detachment of the cell monolayer. If any of these situations occur, it is not realistic to proceed with data analysis until the problem is corrected. The best policy is to attempt to standardize all types of cultures used for analysis and to avoid using any cultures that display atypical characteristics.

  14. TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells.

    PubMed

    Eisele, Günter; Wischhusen, Jörg; Mittelbronn, Michel; Meyermann, Richard; Waldhauer, Inja; Steinle, Alexander; Weller, Michael; Friese, Manuel A

    2006-09-01

    NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.

  15. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

  16. Macrophages help NK cells to attack tumor cells by stimulatory NKG2D ligand but protect themselves from NK killing by inhibitory ligand Qa-1.

    PubMed

    Zhou, Zhixia; Zhang, Cai; Zhang, Jian; Tian, Zhigang

    2012-01-01

    Natural killer (NK) cells and their crosstalk with other immune cells are important for innate immunity against tumor. To explore the role of the interaction between NK cells and macrophages in the regulation of anti-tumor activities of NK cells, we here demonstrate that poly I:C-treated macrophages increased NK cell-mediated cytotoxicity against target tumor cells in NKG2D-dependent manner. In addition, IL-15, IL-18, and IFN-β secreted by poly I:C-treated macrophages are also involved in NKG2D expression and NK cell activation. Interestingly, the increase in expression of NKG2D ligands on macrophages induced a highly NK cell-mediated cytotoxicity against tumor cells, but not against macrophages themselves. Notably, a high expression level of Qa-1, a NKG2A ligand, on macrophages may contribute to such protection of macrophages from NK cell-mediated killing. Furthermore, Qa-1 or NKG2A knockdown and Qa-1 antibody blockade caused the macrophages to be sensitive to NK cytolysis. These results suggested that macrophages may activate NK cells to attack tumor by NKG2D recognition whereas macrophages protect themselves from NK lysis via preferential expression of Qa-1.

  17. Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function.

    PubMed

    Morvan, Maelig G; Champsaur, Marine; Reizis, Boris; Lanier, Lewis L

    2017-05-01

    To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand-expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk.

  18. 3D Cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer.

    PubMed

    Chambers, Karen F; Mosaad, Eman M O; Russell, Pamela J; Clements, Judith A; Doran, Michael R

    2014-01-01

    Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.

  19. Exploiting natural killer group 2D receptors for CAR T-cell therapy.

    PubMed

    Demoulin, Benjamin; Cook, W James; Murad, Joana; Graber, David J; Sentman, Marie-Louise; Lonez, Caroline; Gilham, David E; Sentman, Charles L; Agaugue, Sophie

    2017-08-01

    Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

  20. Nonrigid Registration of 2-D and 3-D Dynamic Cell Nuclei Images for Improved Classification of Subcellular Particle Motion

    PubMed Central

    Kim, Il-Han; Chen, Yi-Chun M.; Spector, David L.; Eils, Roland; Rohr, Karl

    2012-01-01

    The observed motion of subcellular particles in fluorescence microscopy image sequences of live cells is generally a superposition of the motion and deformation of the cell and the motion of the particles. Decoupling the two types of movements to enable accurate classification of the particle motion requires the application of registration algorithms. We have developed an intensity-based approach for nonrigid registration of multi-channel microscopy image sequences of cell nuclei. First, based on 3-D synthetic images we demonstrate that cell nucleus deformations change the observed motion types of particles and that our approach allows to recover the original motion. Second, we have successfully applied our approach to register 2-D and 3-D real microscopy image sequences. A quantitative experimental comparison with previous approaches for nonrigid registration of cell microscopy has also been performed. PMID:20840894

  1. Rapid authentication of different ages of tissue-cultured and wild Dendrobium huoshanense as well as wild Dendrobium henanense using FTIR and 2D-COS IR

    NASA Astrophysics Data System (ADS)

    Chen, Nai-Dong; Chen, Nai-Fu; Li, Jun; Cao, Cai-Yun; Wang, Jin-Mei

    2015-12-01

    The accumulating of pharmaceutical chemicals in medicinal plants would greatly be affected by their ages and establishing a fast quality-identification method to evaluate the similarity of medicinal herbs at different cultivated ages is a critical step for assurance of quality and safety in the TCM industry. In this work, tri-step IR macro-fingerprinting and 2D-COS IR spectrum techniques combined with statistical pattern recognition were applied for discrimination and similarity evaluation of different ages of tissue-cultured and wild Dendrobium huoshanense C. Z. Tang et S. J. Cheng as well as Dendrobium henanense J.L.Lu et L.X Gao. Both tissue-cultured and wild D. huoshanense were easily differentiated from D. henanense by FTIR and SD-IR spectra, while it's quite difficult to discriminate different cultivated years of the three investigated Dendrobiums. In 2D-COS IR spectra, 1-5 auto-peaks with different indensity and positions were located in the region 1160-1030 cm-1 of the twelve Dendrobium samples and thus could be used to identify Dendrobium samples at different ages. Principle component analysis (PCA) of synchronous 2D-COS data showed that the twelve samples were effectively identified and evaluated. The results indicated that the tri-step infrared macro-fingerprinting combined with PCA method was suitable to differentiate the cultivated ages of Dendrobiums with species and orgins rapidly and nondestructively.

  2. DNA demethylation and histone H3K9 acetylation determine the active transcription of the NKG2D gene in human CD8+ T and NK cells

    PubMed Central

    Fernández-Sánchez, Alba; Baragaño Raneros, Aroa; Carvajal Palao, Reyes; Sanz, Ana B.; Ortiz, Alberto; Ortega, Francisco; Suárez-Álvarez, Beatriz; López-Larrea, Carlos

    2013-01-01

    The human activating receptor NKG2D is mainly expressed by NK, NKT, γδ T and CD8+ T cells and, under certain conditions, by CD4+ T cells. This receptor recognizes a diverse family of ligands (MICA, MICB and ULBPs 1–6) leading to the activation of effector cells and triggering the lysis of target cells. The NKG2D receptor-ligand system plays an important role in the immune response to infections, tumors, transplanted graft and autoantigens. Elucidation of the regulatory mechanisms of NKG2D is therefore essential for therapeutic purposes. In this study, we speculate whether epigenetic mechanisms, such as DNA methylation and histone acetylation, participate in NKG2D gene regulation in T lymphocytes and NK cells. DNA methylation in the NKG2D gene was observed in CD4+ T lymphocytes and T cell lines (Jurkat and HUT78), while this gene was unmethylated in NKG2D-positive cells (CD8+ T lymphocytes, NK cells and NKL cell line) and associated with high levels of histone H3 lysine 9 acetylation (H3K9Ac). Treatment with the histone acetyltransferase (HAT) inhibitor curcumin reduces H3K9Ac levels in the NKG2D gene, downregulates NKG2D transcription and leads to a marked reduction in the lytic capacity of NKG2D-mediated NKL cells. These findings suggest that differential NKG2D expression in the different cell subsets is regulated by epigenetic mechanisms and that its modulation by epigenetic treatments might provide a new strategy for treating several pathologies. PMID:23235109

  3. The human NKG2D ligand ULBP2 can be expressed at the cell surface with or without a GPI anchor and both forms can activate NK cells

    PubMed Central

    Fernández-Messina, Lola; Ashiru, Omodele; Agüera-González, Sonia; Reyburn, Hugh T.; Valés-Gómez, Mar

    2011-01-01

    The activating immune receptor NKG2D binds to several stress-induced ligands that are structurally different. MHC-class-I-related chain (MIC) A/B molecules have a transmembrane domain, whereas most UL16 binding proteins (ULBPs) are glycosylphosphatidylinositol (GPI)-linked molecules. The significance of this variability in membrane anchors is unclear. Here, we demonstrate that ULBP2, but not ULBP1 or ULBP3, can reach the cell surface without the GPI modification. Several proteins are expressed at the cell surface as both transmembrane and GPI-linked molecules, either via alternative splicing or by the expression of linked genes. However, to our knowledge, ULBP2 is the first single mammalian cDNA that can be expressed as either a transmembrane or a GPI-anchored protein. The rate of maturation and the levels of cell surface expression of the non-GPI-linked form were lower than those of the GPI-linked ULBP2. Nonetheless, non-GPI ULBP2 was recognised by NKG2D and triggered NK cell cytotoxicity. These data show that differences in membrane attachment by NKG2D ligands are more important for regulation of their surface expression than for cytotoxic recognition by NKG2D and emphasise that detailed characterisation of the cell biology of individual NKG2D ligands will be necessary to allow targeted modulation of this system. PMID:21224393

  4. Efficient ambient-air-stable solar cells with 2D-3D heterostructured butylammonium-caesium-formamidinium lead halide perovskites

    NASA Astrophysics Data System (ADS)

    Wang, Zhiping; Lin, Qianqian; Chmiel, Francis P.; Sakai, Nobuya; Herz, Laura M.; Snaith, Henry J.

    2017-09-01

    Perovskite solar cells are remarkably efficient; however, they are prone to degradation in water, oxygen and ultraviolet light. Cation engineering in 3D perovskite absorbers has led to reduced degradation. Alternatively, 2D Ruddlesden-Popper layered perovskites exhibit improved stability, but have not delivered efficient solar cells so far. Here, we introduce n-butylammonium cations into a mixed-cation lead mixed-halide FA0.83Cs0.17Pb(IyBr1‑y)3 3D perovskite. We observe the formation of 2D perovskite platelets, interspersed between highly orientated 3D perovskite grains, which suppress non-radiative charge recombination. We investigate the relationship between thin-film composition, crystal alignment and device performance. Solar cells with an optimal butylammonium content exhibit average stabilized power conversion efficiency of 17.5 ± 1.3% with a 1.61-eV-bandgap perovskite and 15.8 ± 0.8% with a 1.72-eV-bandgap perovskite. The stability under simulated sunlight is also enhanced. Cells sustain 80% of their 'post burn-in' efficiency after 1,000 h in air, and close to 4,000 h when encapsulated.

  5. Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D.

    PubMed

    Gröschel, Carina; Hübscher, Daniela; Nolte, Jessica; Monecke, Sebastian; Sasse, André; Elsner, Leslie; Paulus, Walter; Trenkwalder, Claudia; Polić, Bojan; Mansouri, Ahmed; Guan, Kaomei; Dressel, Ralf

    2017-01-01

    Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1(-)(/)(-) mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability

  6. High density cell culture system

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor)

    1994-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  7. Phase coding by grid cells in unconstrained environments: Two-dimensional (2D) phase precession

    PubMed Central

    Climer, Jason R.; Newman, Ehren L.; Hasselmo, Michael E.

    2014-01-01

    Action potential timing is thought to play a critical role in neural representation. For example, theta phase precession is a robust phenomenon exhibited by spatial cells of the rat entorhinal-hippocampal circuit. In phase precession, the time a neuron fires relative to the phase of theta rhythm (6-10Hz) oscillations in the local field potential reduces uncertainty about the position of the animal. This relationship between neural firing and behavior has made precession an important constraint for hypothetical mechanisms of temporal coding. However, challenges exist in identifying what regulates the spike timing of these cells. We have developed novel analytical techniques for mapping between behavior and neural firing that provide sufficient sensitivity to examine features of grid cell phase coding in open environments. Here, we show robust, omnidirectional phase precession by entorhinal grid cells in openfield enclosures. We present evidence that full phase precession persists regardless of how close the animal comes to the center of a firing field. We found many conjunctive grid cells, previously thought to be phase locked, also exhibit phase coding. However, we were unable to detect directional or field specific phase coding predicted by some variants of models. Finally, we present data that suggests bursting of layer II grid cells contributes to the bimodality of phase precession. We discuss implications of these observations for models of temporal coding and propose the utility of these techniques in other domains where behavior is aligned to neural spiking. PMID:23718553

  8. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    PubMed

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  9. [Detection of hybrid DQ molecules by the use of T cell clone and 2D-gel analyses].

    PubMed

    Hawkin, S

    1986-11-01

    The HLA-D region incorporates three subregions, DR, DQ and DP, encoding for three sets of Ia molecules. Whereas DR antigens consist of a constant alpha chain and an extremely polymorphic beta chain, both of alpha and beta chain of DQ antigens show moderate polymorphism. This indicated us the existence of hybrid HLA-DQ molecules in HLA-D heterozygous cells, resulting from the association of an alpha chain and a beta chain encoded by genes located on the two separate haplotypes. In this report, hybrid DQ antigens were demonstrated by using cytotoxic T cell-clone. A cytotoxic T cell clone, which was generated by mixed lymphocyte reaction against a lymphoblastoid B cell line, EBV-Fuk (HLA-DR1/4, DQw1/Wa), recognized only heterogenous lymphoblastoid B cell lines (HLA-DR1/4, DQw1/Wa). Cytotoxic T cell clone, however, didn't react with B cell lines which are homozygous for HLA-DR1, DQw1 or DR4/DQWa. This suggests the T cell clone recognized the hybrid DQ molecules expressed only on heterozygous cell lines. Further confirmation was obtained by inhibition test using monoclonal antibody and biochemically by 2-D gel analyses. Biological significance of hybrid DQ antigens were discussed.

  10. Principles of cancer cell culture.

    PubMed

    Cree, Ian A

    2011-01-01

    The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory.

  11. 2D modeling of silicon based thin film dual and triple junction solar cells

    NASA Astrophysics Data System (ADS)

    Xiao, Y. G.; Uehara, K.; Lestrade, M.; Li, Z. Q.; Li, Z. M. S.

    2009-08-01

    Based on Crosslight APSYS, thin film amorphous Si (a-Si:H)/microcrystalline (μc-Si) dual-junction (DJ) and a- Si:H/amorphous SiGe:H (a-SiGe:H)/μc-Si triple-junction (TJ) solar cells are modeled. Basic physical quantities like band diagrams, optical absorption and generation are obtained. Quantum efficiency and I-V curves for individual junctions are presented for current matching analyses. The whole DJ and TJ cell I-V curves are also presented and the results are discussed with respect to the top surface ZnO:Al TCO layer affinity. The interface texture effect is modeled with FDTD (finite difference time domain) module and results for top junction are presented. The modeling results give possible clues to achieve high efficiency for DJ and TJ thin film solar cells.

  12. 2D and 3D fault basis for fuel cell diagnosis by external magnetic field measurements

    NASA Astrophysics Data System (ADS)

    Ifrek, Lyes; Cauffet, Gilles; Chadebec, Olivier; Bultel, Yann; Rosini, Sébastien; Rouveyre, Luc

    2017-07-01

    An original approach used for the identification of faults in fuel cell stacks is presented. It is based on the 3D reconstruction of the current density from external magnetic field measurements which is an ill-posed magnetostatic linear inverse problem. A suitable and original current density and magnetic field basis are proposed in order to define both local and global faults on a fuel cell stack. The inverse problem is regularized by truncated singular value decomposition (SVD) to ensure the uniqueness of the solution. Contribution to the topical issue "Electrical Engineering Symposium (SGE 2016)", edited by Adel Razek

  13. Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog, EB1089

    NASA Technical Reports Server (NTRS)

    Narayanan, R.; Smith, C. L.; Weigel, N. L.

    2002-01-01

    Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect. Copyright 2002 Elsevier Science Inc.

  14. Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog, EB1089.

    PubMed

    Narayanan, R; Smith, C L; Weigel, N L

    2002-09-01

    Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect. Copyright 2002 Elsevier Science Inc.

  15. Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog, EB1089

    NASA Technical Reports Server (NTRS)

    Narayanan, R.; Smith, C. L.; Weigel, N. L.

    2002-01-01

    Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect. Copyright 2002 Elsevier Science Inc.

  16. Cytotoxicity Profile of Endodontic Sealers Provided by 3D Cell Culture Experimental Model.

    PubMed

    Silva, Emmanuel João Nogueira Leal; Carvalho, Nancy Kudsi de; Ronconi, Carina Taboada; De-Deus, Gustavo; Zuolo, Mario Luis; Zaia, Alexandre Augusto

    2016-01-01

    The aim of the present study was to evaluate the cytotoxic effects of five endodontic sealers (AH Plus, Endomethasone N, EndoSequence BC, MTA Fillapex and Pulp Canal Sealer EWT) using a three-dimensional (3D) cell culture model. A conventional bi-dimensional (2D) cell culture model was used as reference technique for comparison. Balb/c 3T3 fibroblasts were cultured in conventional bi-dimensional cell culture and in rat-tail collagen type I three-dimensional cell culture models. Then, both cell cultures were incubated with elutes of freshly mixed endodontic sealers for 24 h. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay (MTT). Data were statistically analyzed using ANOVA and the Tukey test at a significance level of p<0.05. All tested sealers exhibited cytotoxic effects; however, cytotoxic effect was culture model- and sealer-dependent. Sealers showed higher cytotoxicity in 2D than in 3D cell culture model (p<0.05). In both conditions, EndoSequence BC showed the lowest cytotoxicity (p<0.05). MTA Fillapex was much more cytotoxic than the other tested endodontic sealers (p<0.05), with the exception of AH Plus in the 2D cell culture model (p>0.05). Endomethasone N and Pulp Canal Sealer EWT showed lower cytotoxic effects than AH Plus in 2D cell culture model (p<0.05); however no statistical differences was observed among these sealers in 3D cell culture model. It may be concluded that cytotoxicity was higher in 2D cell culture compared to 3D cell culture. EndoSequence BC sealer exhibited the highest cytocompatibility and MTA Fillapex the lowest cytocompatibility.

  17. Fc-optimized NKG2D-Fc constructs induce NK cell antibody-dependent cellular cytotoxicity against breast cancer cells independently of HER2/neu expression status.

    PubMed

    Raab, Stefanie; Steinbacher, Julia; Schmiedel, Benjamin J; Kousis, Philaretos C; Steinle, Alexander; Jung, Gundram; Grosse-Hovest, Ludger; Salih, Helmut R

    2014-10-15

    The ability of NK cells to mediate Ab-dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of antitumor Abs, including trastuzumab, which is approved for the treatment of breast cancer with HER2/neu overexpression. Notably, only ∼25% of breast cancer patients overexpress HER2/neu. Moreover, HER2/neu is expressed on healthy cells, and trastuzumab application is associated with side effects. In contrast, the ligands of the activating immunoreceptor NKG2D (NKG2DL) are selectively expressed on malignant cells. In this study, we took advantage of the tumor-associated expression of NKG2DL by using them as target Ags for NKG2D-IgG1 fusion proteins optimized by amino acid exchange S239D/I332E in their Fc part. Compared to constructs with wild-type Fc parts, fusion proteins carrying the S239D/I332E modification (NKG2D-Fc-ADCC) mediated highly enhanced degranulation, ADCC, and IFN-γ production of NK cells in response to breast cancer cells. NKG2D-Fc-ADCC substantially enhanced NK reactivity also against HER2/neu-low targets that were unaffected by trastuzumab, as both compounds mediated their immunostimulatory effects in strict dependence of target Ag expression levels. Thus, in line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16, NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.

  18. Three-dimensional cell culture models for investigating human viruses.

    PubMed

    He, Bing; Chen, Guomin; Zeng, Yi

    2016-10-01

    Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

  19. Decreased percentage of NKG2D+NK cells in patients with incident onset of Type 1 Diabetes.

    PubMed

    Zhang, Yupan; Wang, Haifeng; Lou, Xiaoqian; Qu, Xiaozhang; Gao, Lichao; Liu, Xiaolei; Li, Man; Guo, Hui; Jiang, Yanfang

    2017-02-01

    Type 1 diabetes mellitus (T1DM) is characterized by absolute insulin deficiency owing to autoimmune destruction of the pancreatic β cells. A significant decrease in natural killer (NK) cells in peripheral blood has been observed in patients with untreated T1DM. In the present study, we aimed to explore the role of NK cells and their subsets in young T1DM patients. A total of 30 children and adolescents with untreated T1DM and 27 healthy controls (HC) were recruited in this study. Flow cytometry analysis indicated that the percentage of peripheral blood CD3-CD56+ NK cells and NK cells subsets (CD56bright, CD56dim and CD56neg), were significantly decreased in the T1DM patients compared to healthy controls. In addition, the percentage of inducible CD107a+ and IFN-γ-secreting NK cells was significantly decreased compared to HC. Interestingly, the percentage of NKG2D+ NK cells negatively correlated with the level of serum TCHOL and TG in T1DM patients. Our data indicate that decreased number and impaired function of NK cells may have a role in the pathogenesis of T1DM.

  20. CD3(+) CD8(+) NKG2D(+) T Lymphocytes Induce Apoptosis and Necroptosis in HLA-negative Cells via FasL-Fas Interaction.

    PubMed

    Ivanova, O K; Sharapova, T N; Romanova, E A; Sashchenko, L P; Yashin, D V

    2017-03-15

    An important problem in cellular immunology is to identify new populations of cytotoxic lymphocytes capable of killing tumor cells that have lost classical components of MHC-machinery and to understand mechanisms of the death of these cells. We have previously found that CD4(+) CD25(+) lymphocytes appear in the lymphokine-activated killer (LAK) cell culture, which carry Tag7 (PGRP-S) and FasL proteins on their surface and can kill Hsp70- and Fas-expressing HLA-negative cells. In this work, we have continued to study the mechanisms of killing of the HLA-negative tumor cells, focusing this time on the CD8+ lymphocytes. We show that after a tumor antigen contact the IL-2 activated CD8+ lymphocytes acquire ability to lyse tumor cells bearing this antigen. However, activation of the CD8+ lymphocytes in the absence of antigen causes appearance of a cytotoxic population of CD8 + NKG2D+ lymphocytes, which are able to lyse HLA-negative cancer cells that have lost the classic mechanism of antigen presentation. These cells recognize the noncanonical MicA antigen on the surface of HLA-negative K562 cells but kill them via the FasL-Fas interaction, as do cytotoxic T lymphocytes. FasL presented on the lymphocyte surface can trigger both apoptosis and necroptosis. Unlike in the case of TNFR1, another cell death receptor, no switching to alternative processes has been observed upon induction of Fas-dependent cell death. It may well be that the apoptotic and necroptotic signals are transduced separately in the latter case, with the ability of FasL(+) lymphocytes to induce necroptosis allowing them to kill tumor cells that escape apoptosis. This article is protected by copyright. All rights reserved.

  1. NKG2D mediates NK cell hyperresponsiveness and influenza-induced pathologies in a mouse model of chronic obstructive pulmonary disease.

    PubMed

    Wortham, Brian W; Eppert, Bryan L; Motz, Greg T; Flury, Jennifer L; Orozco-Levi, Mauricio; Hoebe, Kasper; Panos, Ralph J; Maxfield, Melissa; Glasser, Stephan W; Senft, Albert P; Raulet, David H; Borchers, Michael T

    2012-05-01

    Chronic obstructive pulmonary disease (COPD) is characterized by peribronchial and perivascular inflammation and largely irreversible airflow obstruction. Acute disease exacerbations, due frequently to viral infections, lead to enhanced disease symptoms and contribute to long-term progression of COPD pathology. Previously, we demonstrated that NK cells from cigarette smoke (CS)-exposed mice exhibit enhanced effector functions in response to stimulating cytokines or TLR ligands. In this article, we show that the activating receptor NKG2D is a key mediator for CS-stimulated NK cell hyperresponsiveness, because CS-exposed NKG2D-deficient mice (Klrk1(-/-)) did not exhibit enhanced effector functions as assessed by cytokine responsiveness. NK cell cytotoxicity against MHC class I-deficient targets was not affected in a COPD model. However, NK cells from CS-exposed mice exhibit greater cytotoxic activity toward cells that express the NKG2D ligand RAET1ε. We also demonstrate that NKG2D-deficient mice exhibit diminished airway damage and reduced inflammation in a model of viral COPD exacerbation, which do not affect viral clearance. Furthermore, adoptive transfer of NKG2D(+) NK cells into CS-exposed, influenza-infected NKG2D-deficient mice recapitulated the phenotypes observed in CS-exposed, influenza-infected wild-type mice. Our findings indicate that NKG2D stimulation during long-term CS exposure is a central pathway in the development of NK cell hyperresponsiveness and influenza-mediated exacerbations of COPD.

  2. Synthesizing 2D MoS2 Nanofins on carbon nanospheres as catalyst support for Proton Exchange Membrane Fuel Cells

    NASA Astrophysics Data System (ADS)

    Hu, Yan; Chua, Daniel H. C.

    2016-06-01

    Highly dense 2D MoS2 fin-like nanostructures on carbon nanospheres were fabricated and formed the main catalyst support structure in the oxygen reduction reaction (ORR) for polymer electrolyte membrane (PEM) fuel cells. These nanofins were observed growing perpendicular to the carbon nanosphere surface in random orientations and high resolution transmission electron microscope confirmed 2D layers. The PEM fuel cell test showed enhanced electrochemical activity with good stability, generating over 8.5 W.mgPt-1 as compared to standard carbon black of 7.4 W.mgPt-1 under normal operating conditions. Electrochemical Impedance Spectroscopy confirmed that the performance improvement is highly due to the excellent water management of the MoS2 lamellar network, which facilitates water retention at low current density and flood prevention at high current density. Reliability test further demonstrated that these nanofins are highly stable in the electrochemical reaction and is an excellent ORR catalyst support.

  3. Synthesizing 2D MoS2 Nanofins on carbon nanospheres as catalyst support for Proton Exchange Membrane Fuel Cells

    PubMed Central

    Hu, Yan; Chua, Daniel H. C.

    2016-01-01

    Highly dense 2D MoS2 fin-like nanostructures on carbon nanospheres were fabricated and formed the main catalyst support structure in the oxygen reduction reaction (ORR) for polymer electrolyte membrane (PEM) fuel cells. These nanofins were observed growing perpendicular to the carbon nanosphere surface in random orientations and high resolution transmission electron microscope confirmed 2D layers. The PEM fuel cell test showed enhanced electrochemical activity with good stability, generating over 8.5 W.mgPt−1 as compared to standard carbon black of 7.4 W.mgPt−1 under normal operating conditions. Electrochemical Impedance Spectroscopy confirmed that the performance improvement is highly due to the excellent water management of the MoS2 lamellar network, which facilitates water retention at low current density and flood prevention at high current density. Reliability test further demonstrated that these nanofins are highly stable in the electrochemical reaction and is an excellent ORR catalyst support. PMID:27302135

  4. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells

    NASA Astrophysics Data System (ADS)

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W.

    2016-08-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, +H, 56Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004 +H P = 0.049 56Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation—implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure.

  5. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells

    PubMed Central

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W.

    2016-01-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, +H, 56Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004; +H P = 0.049; 56Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation—implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure. PMID:27539227

  6. A functional polymorphism in the NKG2D gene modulates NK-cell cytotoxicity and is associated with susceptibility to Human Papilloma Virus-related cancers.

    PubMed

    Espinoza, J Luis; Nguyen, Viet H; Ichimura, Hiroshi; Pham, Trang T T; Nguyen, Cuong H; Pham, Thuc V; Elbadry, Mahmoud I; Yoshioka, Katsuji; Tanaka, Junji; Trung, Ly Q; Takami, Akiyoshi; Nakao, Shinji

    2016-12-20

    Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide and is etiologically linked to several cancers, including cervical and genital cancers. NKG2D, an activating receptor expressed by NK cells, plays an important role in cancer immune-surveillance. We analyzed the impact of a NKG2D gene variant, rs1049174, on the incidence of HPV-related cancers in Vietnamese patients and utilized various molecular approaches to elucidate the mechanisms of NKG2D receptor regulation by rs1049174. In a group of 123 patients with HPV+ anogenital cancers, the low cytotoxicity allele LNK was significantly associated with increased cancer susceptibility (p = 0.016). Similar results were also observed in a group of 153 women with cervical cancer (p = 0.05). In functional studies, NK cells from individuals with LNK genotype showed a lower NKG2D expression and displayed less efficient NKG2D-mediated functions than NK cells with HNK genotype. Notably, the rs1049174 variant occurs within a targeting site for miR-1245, a negative regulator of NKG2D expression. Compared with the higher cytotoxicity allele HNK, the LNK allele was more efficiently targeted by miR-1245 and thus determined lower NKG2D expression in NK cells with the LNK genotype. The NKG2D variants may influence cancer immunosurveillance and thus determine susceptibility to various malignancies, including HPV-induced cancers.

  7. A functional polymorphism in the NKG2D gene modulates NK-cell cytotoxicity and is associated with susceptibility to Human Papilloma Virus-related cancers

    PubMed Central

    Espinoza, J. Luis; Nguyen, Viet H.; Ichimura, Hiroshi; Pham, Trang T. T.; Nguyen, Cuong H.; Pham, Thuc V.; Elbadry, Mahmoud I.; Yoshioka, Katsuji; Tanaka, Junji; Trung, Ly Q.; Takami, Akiyoshi; Nakao, Shinji

    2016-01-01

    Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide and is etiologically linked to several cancers, including cervical and genital cancers. NKG2D, an activating receptor expressed by NK cells, plays an important role in cancer immune-surveillance. We analyzed the impact of a NKG2D gene variant, rs1049174, on the incidence of HPV-related cancers in Vietnamese patients and utilized various molecular approaches to elucidate the mechanisms of NKG2D receptor regulation by rs1049174. In a group of 123 patients with HPV+ anogenital cancers, the low cytotoxicity allele LNK was significantly associated with increased cancer susceptibility (p = 0.016). Similar results were also observed in a group of 153 women with cervical cancer (p = 0.05). In functional studies, NK cells from individuals with LNK genotype showed a lower NKG2D expression and displayed less efficient NKG2D-mediated functions than NK cells with HNK genotype. Notably, the rs1049174 variant occurs within a targeting site for miR-1245, a negative regulator of NKG2D expression. Compared with the higher cytotoxicity allele HNK, the LNK allele was more efficiently targeted by miR-1245 and thus determined lower NKG2D expression in NK cells with the LNK genotype. The NKG2D variants may influence cancer immunosurveillance and thus determine susceptibility to various malignancies, including HPV-induced cancers. PMID:27995954

  8. Cell culture purity issues and DFAT cells

    SciTech Connect

    Wei, Shengjuan; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  9. Real-time monitoring of 3D cell culture using a 3D capacitance biosensor.

    PubMed

    Lee, Sun-Mi; Han, Nalae; Lee, Rimi; Choi, In-Hong; Park, Yong-Beom; Shin, Jeon-Soo; Yoo, Kyung-Hwa

    2016-03-15

    Three-dimensional (3D) cell cultures have recently received attention because they represent a more physiologically relevant environment compared to conventional two-dimensional (2D) cell cultures. However, 2D-based imaging techniques or cell sensors are insufficient for real-time monitoring of cellular behavior in 3D cell culture. Here, we report investigations conducted with a 3D capacitance cell sensor consisting of vertically aligned pairs of electrodes. When GFP-expressing human breast cancer cells (GFP-MCF-7) encapsulated in alginate hydrogel were cultured in a 3D cell culture system, cellular activities, such as cell proliferation and apoptosis at different heights, could be monitored non-invasively and in real-time by measuring the change in capacitance with the 3D capacitance sensor. Moreover, we were able to monitor cell migration of human mesenchymal stem cells (hMSCs) with our 3D capacitance sensor.

  10. A 2D Particle in Cell model for ion extraction and focusing in electrostatic accelerators.

    PubMed

    Veltri, P; Cavenago, M; Serianni, G

    2014-02-01

    Negative ions are fundamental to produce intense and high energy neutral beams used to heat the plasma in fusion devices. The processes regulating the ion extraction involve the formation of a sheath on a scale comparable to the Debye length of the plasma. On the other hand, the ion acceleration as a beam is obtained on distances greater than λD. The paper presents a model for both the phases of ion extraction and acceleration of the ions and its implementation in a numerical code. The space charge of particles is deposited following usual Particle in Cell codes technique, while the field is solved with finite element methods. Some hypotheses on the beam plasma transition are described, allowing to model both regions at the same time. The code was tested with the geometry of the NIO1 negative ions source, and the results are compared with existing ray tracing codes and discussed.

  11. A 2D Particle in Cell model for ion extraction and focusing in electrostatic accelerators

    SciTech Connect

    Veltri, P. Serianni, G.; Cavenago, M.

    2014-02-15

    Negative ions are fundamental to produce intense and high energy neutral beams used to heat the plasma in fusion devices. The processes regulating the ion extraction involve the formation of a sheath on a scale comparable to the Debye length of the plasma. On the other hand, the ion acceleration as a beam is obtained on distances greater than λ{sub D}. The paper presents a model for both the phases of ion extraction and acceleration of the ions and its implementation in a numerical code. The space charge of particles is deposited following usual Particle in Cell codes technique, while the field is solved with finite element methods. Some hypotheses on the beam plasma transition are described, allowing to model both regions at the same time. The code was tested with the geometry of the NIO1 negative ions source, and the results are compared with existing ray tracing codes and discussed.

  12. Resistance to Cytarabine Induces the Up-regulation of NKG2D Ligands and Enhances Natural Killer Cell Lysis of Leukemic Cells1

    PubMed Central

    Ogbomo, Henry; Michaelis, Martin; Klassert, Denise; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2008-01-01

    Prolonged treatment of leukemic cells with chemotherapeutic agents frequently results in development of drug resistance. Moreover, selection of drug-resistant cell populations may be associated with changes in malignant properties such as proliferation rate, invasiveness, and immunogenicity. In the present study, the sensitivity of cytarabine (1-β-d-arabinofuranosylcytosine, araC)-resistant and parental human leukemic cell lines (T-lymphoid H9 and acute T-lymphoblastic leukemia Molt-4) to natural killer (NK) cell-mediated killing was investigated. The results obtained demonstrate that araC-resistant H9 and Molt-4 (H9rARAC100 and Molt-4rARAC100) cell lines are more sensitive to NK cell-mediated lysis than their respective parental cell lines. This increased sensitivity was associated with a higher surface expression of ligands for the NK cell-activating receptor NKG2D, notably UL16 binding protein-2 (ULBP-2) and ULBP-3 in H9rARAC100 and Molt-4rARAC100 cell lines. Blocking ULBP-2 and ULBP-3 or NKG2D with monoclonal antibody completely abrogated NK cell lysis. Constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not pAKT was higher in araC-resistant cells than in parental cell lines. Inhibition of ERK using ERK inhibitor PD98059 decreased both ULBP-2/ULBP-3 expression and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in H9 parental cells resulted in increased ULBP-2/ULBP-3 expression and enhanced NK cell lysis. These results demonstrate that increased sensitivity of araC-resistant leukemic cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. PMID:19048119

  13. Histochemical examination of adipose derived stem cells combined with β-TCP for bone defects restoration under systemic administration of 1α,25(OH)2D3.

    PubMed

    Feng, Wei; Lv, Shengyu; Cui, Jian; Han, Xiuchun; Du, Juan; Sun, Jing; Wang, Kefeng; Wang, Zhenming; Lu, Xiong; Guo, Jie; Oda, Kimimitsu; Amizuka, Norio; Xu, Xin; Li, Minqi

    2015-09-01

    The purpose of this study was to evaluate the effects of osteogenic differentiated adipose-derived stem cell (ADSC) loaded beta-tricalcium phosphate (β-TCP) in the restoration of bone defects under intraperitoneal administration of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3). ADSCs were isolated from the fat tissue of 8 week old Wister rats and co-cultured with β-TCP for 21 days under osteogenic induction. Then the ADSC-β-TCP complexes were implanted into bone defects in the femora of rats. 1α,25(OH)2D3 (VD) or normal saline (NS) was administrated intraperitoneally every other day after the surgery. Femora were harvested at day 7, day 14 and day 28 post-surgery. There were 4 groups for all specimens: β-TCP-NS group; β-TCP-ADSC-NS group; β-TCP-VD group and β-TCP-ADSC-VD group. Alkaline phosphatase (ALP) was up-regulated obviously in ADSC groups compared with non-ADSC groups at day 7, day 14 and day 28, although high expression of runt-related transcription factor 2 (RUNX2) was only seen at day 7. Furthermore, the number of TRAP-positive osteoclasts and the expression of cathepsin K (CK) were significantly decreased in VD groups compared with non-VD groups at day 7 and day 14. As a most significant finding, the β-TCP-ADSC-VD group showed the highest BV/TV ratio compared with the other three groups at day 28. Taken together, ADSC-loaded β-TCP under the administration of 1α,25(OH)2D3 made a promising therapy for bone defects restoration.

  14. 2D ratiometric fluorescent pH sensor for tracking of cells proliferation and metabolism.

    PubMed

    Ma, Jun; Ding, Changqin; Zhou, Jie; Tian, Yang

    2015-08-15

    Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved.

  15. Seeing through the trick of cancer cells via 2D gels.

    PubMed

    Mao, Lei

    2013-12-01

    The advancement of modern therapy concepts has dramatically extended the postsurvival rates of patients with malignant gastric cancer. However, a remaining setback is the drug resistance of recurrent cancer, which casts a dark shadow over disease prognosis. The original work of Klein et al. [Proteomics Clin. Appl. 2013, 7, 813-824] has outlined a rational experimental approach to decipher the mechanistic pathway of cancer drug resistance by proteomic approach. They used gel-based comparative proteomics to analyze the nuclear proteome of a human gastric cancer cell line (AGS) with and without inactivation of hypoxia-inducible factor 1 (HIF-1), a transcription factor and master regulator of hypoxia adaptation. Using the classical 2DE-MS approach, these researchers observed 163 HIF-1 responsive proteins, among which over half of them could be confidently identified by MS. From this large dataset, the authors proposed an enhanced nuclear translocation of some proteasomal proteins upon inactivation of HIF-1. Overall, this work appropriately used proteomics as a hypothesis-free, top-down approach to dissect imperative clinical problems.

  16. A Parallel 2D Numerical Simulation of Tumor Cells Necrosis by Local Hyperthermia

    NASA Astrophysics Data System (ADS)

    Reis, R. F.; Loureiro, F. S.; Lobosco, M.

    2014-03-01

    Hyperthermia has been widely used in cancer treatment to destroy tumors. The main idea of the hyperthermia is to heat a specific region like a tumor so that above a threshold temperature the tumor cells are destroyed. This can be accomplished by many heat supply techniques and the use of magnetic nanoparticles that generate heat when an alternating magnetic field is applied has emerged as a promise technique. In the present paper, the Pennes bioheat transfer equation is adopted to model the thermal tumor ablation in the context of magnetic nanoparticles. Numerical simulations are carried out considering different injection sites for the nanoparticles in an attempt to achieve better hyperthermia conditions. Explicit finite difference method is employed to solve the equations. However, a large amount of computation is required for this purpose. Therefore, this work also presents an initial attempt to improve performance using OpenMP, a parallel programming API. Experimental results were quite encouraging: speedups around 35 were obtained on a 64-core machine.

  17. Development of 2D dynamic model for hydrogen-fed and methane-fed solid oxide fuel cells

    NASA Astrophysics Data System (ADS)

    Luo, X. J.; Fong, K. F.

    2016-10-01

    A new two-dimensional (2D) dynamic model is developed in Fortran to study the mass and energy transport, the velocity field and the electrochemical phenomena of high-temperature solid oxide fuel cell (SOFC). The key feature of this model is that gas properties, reaction heat, open circuit voltage, ohmic voltage and exchange current density are temperature-dependent. Based on this, the change of gas temperature and related characteristics can be evaluated in this study. The transient performances of SOFC, like heat-up and start-up processes, are therefore assessed accordingly. In this 2D dynamic SOFC model, chemical and electrochemical reaction, flow field, mass and energy transfer models are coupled in order to determine the current density, the mass fraction and the temperature of gas species. Mass, momentum and energy balance equations are discretized by finite difference method. Performance evaluation in current density, electrical efficiency and overall efficiency is conducted for the effects of different operating parameters in SOFC. The present model can serve as a valuable tool for in-depth performance evaluation of other design and operating parameters of SOFC unit, as well as further dynamic simulation and optimization of SOFC as a prime mover in cogeneration or trigeneration system.

  18. Physical enviroment of 2-D animal cell aggregates formed in a short pathlength ultrasound standing wave trap.

    PubMed

    Bazou, Despina; Kuznetsova, Larisa A; Coakley, W Terence

    2005-03-01

    2-D mammalian cell aggregates can be formed and levitated in a 1.5 MHz single half wavelength ultrasound standing wave trap. The physical environment of cells in such a trap has been examined. Attention was paid to parameters such as temperature, acoustic streaming, cavitation and intercellular forces. The extent to which these factors might be intrusive to a neural cell aggregate levitated in the trap was evaluated. Neural cells were exposed to ultrasound at a pressure amplitude of 0.54 MPa for 30 s; a small aggregate had been formed at the center of the trap. The pressure amplitude was then decreased to 0.27 MPa for 2 min, at which level the aggregation process continued at a slower rate. The pressure amplitude was then decreased to 0.06 MPa for 1 h. Temperature measurements that were conducted in situ with a 200 microm thermocouple over a 30 min period showed that the maximum temperature rise was less than 0.5 K. Acoustic streaming was measured by the particle image velocimetry method (PIV). It was shown that the hydrodynamic stress imposed on cells by acoustic streaming is less than that imposed by gentle preparative centrifugation procedures. Acoustic spectrum analysis showed that cavitation activity does not occur in the cell suspensions sonicated at the above pressures. White noise was detected only at a pressure amplitude of 1.96 MPa. Finally, it was shown that the attractive acoustic force between ultrasonically agglomerated cells is small compared with the normal attractive van der Waals force that operates at close cell surface separations. It is concluded that the standing wave trap operates only to concentrate cells locally, as in tissue, and does not modify the in vitro expression of surface receptor interactions.

  19. Expression and Function of NKG2D Is Impaired in CD8+ T Cells of Chronically HIV-1-Infected Patients Without ART.

    PubMed

    Giuliani, Erica; Vassena, Lia; Desimio, Maria Giovanna; Buonomini, Anna Rita; Malagnino, Vincenzo; Andreoni, Massimo; Doria, Margherita

    2015-12-01

    Increasing line of evidence indicates that the NKG2D-activating receptor plays a relevant role in the effector functions of cytotoxic lymphocytes. In this study, we investigated the expression and function of NKG2D in CD8⁺ T cells from chronically HIV-1-infected patients with or without antiretroviral therapy (ART). We measured by flow cytometry the expression of NKG2D on CD8⁺ T-cell subsets of ART-naive and ART patients as well as seronegative healthy subjects (HIV-neg). An intrapatient analysis before and after ART initiation was also performed. Results were correlated with viral load, CD4⁺ T-cell counts, markers of immune activation (CD38, sCD14), and soluble NKG2D ligands (sMICA and sULBP2). The function of NKG2D on CD8⁺ T cell cytotoxicity was tested by ex vivo degranulation assays. We showed that NKG2D was downregulated on all CD8⁺ T-cell subsets of ART-naive patients. The expression of NKG2D on CD8⁺ T cells inversely correlated with viral load and CD38 expression but not with plasma levels of sMICA and sULBP2. Importantly, we found that NKG2D-mediated costimulation of CD8⁺ T-cell lytic activity was strongly reduced in ART-naive patients if compared with HIV-neg and ART subjects. Finally, intrapatient analysis demonstrated that effective anti-HIV-1 therapy restores NKG2D expression and NKG2D-induced cytotoxicity by CD8⁺ T cells. These data underscore that NKG2D downregulation contributes to impaired CD8⁺ T-cell responses in untreated HIV-1 infection and have implications for monitoring immune functions and response to treatments, and for the development of novel anti-HIV-1 strategies combining ART with drugs that stimulate NKG2D expression and function.

  20. Natural killer cells recognize friend retrovirus-infected erythroid progenitor cells through NKG2D-RAE-1 interactions In Vivo.

    PubMed

    Ogawa, Tatsuya; Tsuji-Kawahara, Sachiyo; Yuasa, Takae; Kinoshita, Saori; Chikaishi, Tomomi; Takamura, Shiki; Matsumura, Haruo; Seya, Tsukasa; Saga, Toshihiko; Miyazawa, Masaaki

    2011-06-01

    Natural killer (NK) cells function as early effector cells in the innate immune defense against viral infections and also participate in the regulation of normal and malignant hematopoiesis. NK cell activities have been associated with early clearance of viremia in experimental simian immunodeficiency virus and clinical human immunodeficiency virus type 1 (HIV-1) infections. We have previously shown that NK cells function as major cytotoxic effector cells in vaccine-induced immune protection against Friend virus (FV)-induced leukemia, and NK cell depletion totally abrogates the above protective immunity. However, how NK cells recognize retrovirus-infected cells remains largely unclear. The present study demonstrates a correlation between the expression of the products of retinoic acid early transcript-1 (RAE-1) genes in target cells and their susceptibility to killing by NK cells isolated from FV-infected animals. This killing was abrogated by antibodies blocking the NKG2D receptor in vitro. Further, the expression of RAE-1 proteins on erythroblast surfaces increased early after FV inoculation, and administration of an RAE-1-blocking antibody resulted in increased spleen infectious centers and exaggerated pathology, indicating that FV-infected erythroid cells are recognized by NK cells mainly through the NKG2D-RAE-1 interactions in vivo. Enhanced retroviral replication due to host gene-targeting resulted in markedly increased RAE-1 expression in the absence of massive erythroid cell proliferation, indicating a direct role of retroviral replication in RAE-1 upregulation.

  1. Cell culture purity issues and DFAT cells.

    PubMed

    Wei, Shengjuan; Bergen, Werner G; Hausman, Gary J; Zan, Linsen; Dodson, Michael V

    2013-04-12

    Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  2. Role of NKG2D, DNAM-1 and natural cytotoxicity receptors in cytotoxicity toward rhabdomyosarcoma cell lines mediated by resting and IL-15-activated human natural killer cells.

    PubMed

    Boerman, Gerharda H; van Ostaijen-ten Dam, Monique M; Kraal, Kathelijne C J M; Santos, Susy J; Ball, Lynne M; Lankester, Arjan C; Schilham, Marco W; Egeler, R Maarten; van Tol, Maarten J D

    2015-05-01

    Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.

  3. Assessing the ability of the 2D Fisher-KPP equation to model cell-sheet wound closure.

    PubMed

    Habbal, Abderrahmane; Barelli, Hélène; Malandain, Grégoire

    2014-06-01

    We address in this paper the ability of the Fisher-KPP equations to render some of the dynamical features of epithelial cell-sheets during wound closure. Our approach is based on nonlinear parameter identification, in a two-dimensional setting, and using advanced 2D image processing of the video acquired sequences. As original contribution, we lead a detailed study of the profiles of the classically used cost functions, and we address the "wound constant speed" assumption, showing that it should be handled with care. We study five MDCK cell monolayer assays in a reference, activated and inhibited migration conditions. Modulo the inherent variability of biological assays, we show that in the assay where migration is not exogeneously activated or inhibited, the wound velocity is constant. The Fisher-KPP equation is able to accurately predict, until the final closure of the wound, the evolution of the wound area, the mean velocity of the cell front, and the time at which the closure occurred. We also show that for activated as well as for inhibited migration assays, many of the cell-sheet dynamics cannot be well captured by the Fisher-KPP model. Finally, we draw some conclusions related to the identified model parameters, and possible utilization of the model. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Application of cell co-culture system to study fat and muscle cells.

    PubMed

    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  5. Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

    PubMed Central

    Bae, Yoo-Jin; Kwon, Yong-Rim; Kim, Hye Joung; Lee, Seok

    2017-01-01

    Background Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. Methods 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. Results AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. Conclusion 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs. PMID:28401097

  6. Effect of cell culture using chitosan membranes on stemness marker genes in mesenchymal stem cells.

    PubMed

    Li, Zhiqiang; Tian, Xiaojun; Yuan, Yan; Song, Zhixiu; Zhang, Lili; Wang, Xia; Li, Tong

    2013-06-01

    Mesenchymal stem cell (MSC) therapy is a promising treatment for diseases of the nervous system. However, MSCs often lose their stemness and homing abilities when cultured in conventional two‑dimensional (2D) systems. Consequently, it is important to explore novel culture methods for MSC-based therapies in clinical practice. To investigate the effect of a cell culture using chitosan membranes on MSCs, the morphology of MSCs cultured using chitosan membranes was observed and the expression of stemness marker genes was analyzed. We demonstrated that MSCs cultured using chitosan membranes form spheroids. Additionally, the expression of stemness marker genes, including Oct4, Sox2 and Nanog, increased significantly when MSCs were cultured using chitosan membranes compared with 2D culture systems. Finally, MSCs cultured using chitosan membranes were found to have an increased potential to differentiate into nerve cells and chrondrocytes. In conclusion, we demonstrated that MSCs cultured on chitosan membranes maintain their stemness and homing abilities. This finding may be further investigated for the development of novel cell-based therapies for diseases involving neuron-like cells and chondrogenesis.

  7. Effects of Hypericum perforatum extract and its main bioactive compounds on the cytotoxicity and expression of CYP1A2 and CYP2D6 in hepatic cells.

    PubMed

    Silva, Sara M; Martinho, Ana; Moreno, Ivo; Silvestre, Samuel; Granadeiro, Luiza Breitenfeld; Alves, Gilberto; Duarte, Ana Paula; Domingues, Fernanda; Gallardo, Eugenia

    2016-01-01

    Hypericum perforatum (H. perforatum) is one of the most used medicinal plants. However, it has been associated with relevant interactions with several drugs. This situation is probably mediated by cytochrome P450 enzymes (CYP450), namely the 1A2 (CYP1A2) and 2D6 (CYP2D6) isoforms This study aims to assess the cytotoxic and CYP1A2 and CYP2D6 inductive and/or inhibitory effects of a H. perforatum extract and its main bioactive components in hepatic cell lines. A MTT proliferation assay was performed in WRL-68, HepG2 and HepaRG cells after exposition to different concentrations of H. perforatum extract, hypericin and hyperforin for 24 and 72 h. Then, a real-time PCR analysis was accomplished after incubating the cells with these products evaluating the relative CYP1A2 and CYP2D6 expression. These products have relevant cytotoxicity at a 10 μM concentration and it was also demonstrated for the first time that H. perforatum can lead to a significant CYP1A2 and CYP2D6 induction in all cell lines. Moreover, hypericin seems to induce CYP1A2 in HepG2 cells and to inhibit its expression in HepaRG cells while hyperforin induced CYP1A2 in HepG2 and in WRL-68 cells. Additionally, hypericin and hyperforin induce CYP2D6 in HepG2 cells but inhibits its expression in HepaRG and in WRL-68 cells. This study not only evidenced that H. perforatum extract and two of its bioactive components can have toxic effects in hepatic cell lines but also emphasized the potential risk of the consumption of H. perforatum with CYP1A2- and CYP2D6-metabolized drugs. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Inhibition of UBE2D3 Expression Attenuates Radiosensitivity of MCF-7 Human Breast Cancer Cells by Increasing hTERT Expression and Activity

    PubMed Central

    Hu, Liu; Li, Fen; Ren, Li; Yu, Haijun; Liu, Yu; Xia, Ling; Lei, Han; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; Zhou, Yunfeng

    2013-01-01

    The known functions of telomerase in tumor cells include replenishing telomeric DNA and maintaining cell immortality. We have previously shown the existence of a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Here we set out to elucidate the molecular mechanisms underlying regulation by telomerase of radiosensitivity in MCF-7 cells. Toward this aim, yeast two-hybrid (Y2H) screening of a human laryngeal squamous cell carcinoma radioresistant (Hep2R) cDNA library was first performed to search for potential hTERT interacting proteins. We identified ubiquitin-conjugating enzyme E2D3 (UBE2D3) as a principle hTERT-interacting protein and validated this association biochemically. ShRNA-mediated inhibition of UBE2D3 expression attenuated MCF-7 radiosensitivity, and induced the accumulation of hTERT and cyclin D1 in these cells. Moreover, down-regulation of UBE2D3 increased hTERT activity and cell proliferation, accelerating G1 to S phase transition in MCF-7 cells. Collectively these findings suggest that UBE2D3 participates in the process of hTERT-mediated radiosensitivity in human breast cancer MCF-7 cells by regulating hTERT and cyclin D1. PMID:23741361

  9. Aseptic technique for cell culture.

    PubMed

    Coté, R J

    2001-05-01

    This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here.

  10. Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition

    PubMed Central

    Jia, Li-Ting; Wang, Hui-Yan; Qin, Yu-Juan; Chen, Lin; Shen, Hong-Fen; Lin, Xiao-Lin; Yang, Jie; Yang, Sheng; Hao, Wei-Chao; Chen, Yan; Xiao, Sheng-Jun; Zhou, Hui-Rong; Lin, Tao-Yan; Chen, Yu-Shuang; Sun, Yan; Yao, Kai-Tai; Xiao, Dong

    2015-01-01

    Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment. PMID:26418951

  11. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  12. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  13. Cell culture compositions

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  14. Silencing NKG2D ligand-targeting miRNAs enhances natural killer cell-mediated cytotoxicity in breast cancer.

    PubMed

    Shen, Jiaying; Pan, Jie; Du, Chengyong; Si, Wengong; Yao, Minya; Xu, Liang; Zheng, Huilin; Xu, Mingjie; Chen, Danni; Wang, Shu; Fu, Peifen; Fan, Weimin

    2017-04-06

    NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DLs). NKG2DLs are expressed on malignant cells and sensitize them to early elimination by cytotoxic lymphocytes. We investigated the clinical importance of NKG2DLs and the mechanism of NKG2DL regulation in breast cancer (BC). Among the NKG2DLs MICA/B and ULBP1/2/3, the expression levels of MICA/B in BC tissues were inversely associated with the Tumor Node Metastasis stage. We first found that the high expression of MICB, but not MICA, was an independent prognostic factor for overall survival in patients with BC. Investigation into the mechanism revealed that a group of microRNAs (miRNAs) belonging to the miR-17-92 cluster, especially miR-20a, decreased the expression of ULBP2 and MICA/B. These miRNAs downregulated the expression of MICA/B by targeting the MICA/B 3'-untranslated region and downregulated ULBP2 by inhibiting the MAPK/ERK signaling pathway. Functional analysis showed that the silencing of NKG2DL-targeting miRNAs in BC cells increased NK cell-mediated cytotoxicity in vitro and inhibited immune escape in vivo. In addition, histone deacetylase inhibitors (HDACis) increased NKG2DL expression in BC cells by inhibiting members of the miR-17-92 cluster. Thus, targeting miRNAs with antisense inhibitors or HDACis may represent a novel approach for increasing the immunogenicity of BC.

  15. Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

    PubMed Central

    2013-01-01

    Background The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8+ cytotoxic T cells and is classically considered absent in CD4+ T cells. However, recent studies have identified a distinctive population of CD4+ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4+ T cell-immunocompromised individuals suggests that CD4+ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4+ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4+NKG2D+ T cell expansion were also measured. Results Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4+ T cells and CD8+ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4+ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4+NKG2D+ T cells was seen in patients with CIN 1, despite the overall levels of CD4+ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4+NKG2D+ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group. Conclusions Taken together, our results suggest that the significant increase within the CD4+NKG2D+ T cell population in CIN 1 patients might be the result of a

  16. Recruitment of activating NK-cell receptors 2B4 and NKG2D to membrane microdomains in mammalian cells is dependent on their transmembrane regions.

    PubMed

    Gütgemann, Stephan A; Sandusky, Mina M; Wingert, Sabine; Claus, Maren; Watzl, Carsten

    2015-04-01

    Membrane microdomains play an important role in the regulation of natural killer (NK) cell activities. These cholesterol-rich membrane domains are enriched at the activating immunological synapse and several activating NK-cell receptors are known to localize to membrane microdomains upon receptor engagement. In contrast, inhibitory receptors do not localize in these specialized membrane domains. In addition, the functional competence of educated NK cells correlates with a confinement of activating receptors in membrane microdomains. However, the molecular basis for this confinement is unknown. Here, we investigate the structural requirements for the recruitment of the human-activating NK-cell receptors NKG2D and 2B4 to detergent-resistant membrane fractions in the murine BA/F3 cell line and in the human NK-cell line NKL. This stimulation-dependent recruitment occurred independently of the intracellular domains of the receptors. However, either interfering with the association between NKG2D and DAP10, or mutating the transmembrane region of 2B4 impacted the recruitment of the receptors to detergent-resistant membrane fractions and modulated the function of 2B4 in NK cells. Our data suggest a potential interaction between the transmembrane region of NK-cell receptors and membrane lipids as a molecular mechanism involved in determining the membrane confinement of activating NK-cell receptors.

  17. Levels of regulatory T cells CD69(+)NKG2D(+)IL-10(+) are increased in patients with autoimmune thyroid disorders.

    PubMed

    Rodríguez-Muñoz, Ana; Vitales-Noyola, Marlen; Ramos-Levi, Ana; Serrano-Somavilla, Ana; González-Amaro, Roberto; Marazuela, Mónica

    2016-03-01

    Regulatory T (Treg) cells play an important role in the pathogenesis of autoimmune thyroid disorders (AITD). New subsets of CD4(+)CD69(+) and CD4(+)NKG2D(+) T lymphocytes that behave as regulatory cells have been recently reported. The role of these immunoregulatory lymphocytes has not been previously explored in AITD. We analyzed by multi-parametric flow cytometry different Treg cell subsets in peripheral blood from 32 patients with AITD and 19 controls, and in thyroid tissue from seven patients. The suppressive activity was measured by an assay of inhibition of lymphocyte activation. We found a significant increased percentage of CD4(+)CD69(+)IL-10(+), CD4(+)CD69(+)NKG2D(+), and CD4(+)CD69(+)IL-10(+)NKG2D(+) cells, in peripheral blood from GD patients compared to controls. The increase in CD4(+)CD69(+)IL-10(+) and CD4(+)CD69(+)IL-10(+)NKG2D(+) T cells was especially remarkable in patients with active Graves' ophthalmopathy (GO), and a significant positive correlation between GO activity and CD4(+)CD69(+)IL-10(+) or CD4(+)CD69(+)IL-10(+)NKG2D(+) cells was also found. In addition, these cells were increased in patients with a more severe and/or prolonged disease. Thyroid from AITD patients showed an increased proportion of CD69(+) regulatory T cells subpopulations compared to autologous peripheral blood. The presence of CD69(+), NKG2D(+), and IL-10(+) cells was confirmed by immunofluorescence microscopy. In vitro functional assays showed that CD69(+) Treg cells exerted an important suppressive effect on the activation of T effector cells in controls, but not in AITD patients. Our findings suggest that the levels of CD69(+) regulatory lymphocytes are increased in AITD patients, but they are apparently unable to down-modulate the autoimmune response and tissue damage.

  18. Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors

    PubMed Central

    Edmondson, Rasheena; Broglie, Jessica Jenkins; Adcock, Audrey F.

    2014-01-01

    Abstract Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In this review, we discuss the characteristics of 3D cell culture systems in comparison to the two-dimensional (2D) monolayer culture, focusing on cell growth conditions, cell proliferation, population, and gene and protein expression profiles. The innovations and development in 3D culture systems for drug discovery over the past 5 years are also reviewed in the article, emphasizing the cellular response to different classes of anticancer drugs, focusing particularly on similarities and differences between 3D and 2D models across the field. The progression and advancement in the application of 3D cell cultures in cell-based biosensors is another focal point of this review. PMID:24831787

  19. 11.4% Efficiency non-fullerene polymer solar cells with trialkylsilyl substituted 2D-conjugated polymer as donor.

    PubMed

    Bin, Haijun; Gao, Liang; Zhang, Zhi-Guo; Yang, Yankang; Zhang, Yindong; Zhang, Chunfeng; Chen, Shanshan; Xue, Lingwei; Yang, Changduk; Xiao, Min; Li, Yongfang

    2016-12-01

    Simutaneously high open circuit voltage and high short circuit current density is a big challenge for achieving high efficiency polymer solar cells due to the excitonic nature of organic semdonductors. Herein, we developed a trialkylsilyl substituted 2D-conjugated polymer with the highest occupied molecular orbital level down-shifted by Si-C bond interaction. The polymer solar cells obtained by pairing this polymer with a non-fullerene acceptor demonstrated a high power conversion efficiency of 11.41% with both high open circuit voltage of 0.94 V and high short circuit current density of 17.32 mA cm(-2) benefitted from the complementary absorption of the donor and acceptor, and the high hole transfer efficiency from acceptor to donor although the highest occupied molecular orbital level difference between the donor and acceptor is only 0.11 eV. The results indicate that the alkylsilyl substitution is an effective way in designing high performance conjugated polymer photovoltaic materials.

  20. 11.4% Efficiency non-fullerene polymer solar cells with trialkylsilyl substituted 2D-conjugated polymer as donor

    PubMed Central

    Bin, Haijun; Gao, Liang; Zhang, Zhi-Guo; Yang, Yankang; Zhang, Yindong; Zhang, Chunfeng; Chen, Shanshan; Xue, Lingwei; Yang, Changduk; Xiao, Min; Li, Yongfang

    2016-01-01

    Simutaneously high open circuit voltage and high short circuit current density is a big challenge for achieving high efficiency polymer solar cells due to the excitonic nature of organic semdonductors. Herein, we developed a trialkylsilyl substituted 2D-conjugated polymer with the highest occupied molecular orbital level down-shifted by Si–C bond interaction. The polymer solar cells obtained by pairing this polymer with a non-fullerene acceptor demonstrated a high power conversion efficiency of 11.41% with both high open circuit voltage of 0.94 V and high short circuit current density of 17.32 mA cm−2 benefitted from the complementary absorption of the donor and acceptor, and the high hole transfer efficiency from acceptor to donor although the highest occupied molecular orbital level difference between the donor and acceptor is only 0.11 eV. The results indicate that the alkylsilyl substitution is an effective way in designing high performance conjugated polymer photovoltaic materials. PMID:27905397

  1. 11.4% Efficiency non-fullerene polymer solar cells with trialkylsilyl substituted 2D-conjugated polymer as donor

    NASA Astrophysics Data System (ADS)

    Bin, Haijun; Gao, Liang; Zhang, Zhi-Guo; Yang, Yankang; Zhang, Yindong; Zhang, Chunfeng; Chen, Shanshan; Xue, Lingwei; Yang, Changduk; Xiao, Min; Li, Yongfang

    2016-12-01

    Simutaneously high open circuit voltage and high short circuit current density is a big challenge for achieving high efficiency polymer solar cells due to the excitonic nature of organic semdonductors. Herein, we developed a trialkylsilyl substituted 2D-conjugated polymer with the highest occupied molecular orbital level down-shifted by Si-C bond interaction. The polymer solar cells obtained by pairing this polymer with a non-fullerene acceptor demonstrated a high power conversion efficiency of 11.41% with both high open circuit voltage of 0.94 V and high short circuit current density of 17.32 mA cm-2 benefitted from the complementary absorption of the donor and acceptor, and the high hole transfer efficiency from acceptor to donor although the highest occupied molecular orbital level difference between the donor and acceptor is only 0.11 eV. The results indicate that the alkylsilyl substitution is an effective way in designing high performance conjugated polymer photovoltaic materials.

  2. 1α,25(OH)2D3 Suppresses the Migration of Ovarian Cancer SKOV-3 Cells through the Inhibition of Epithelial–Mesenchymal Transition

    PubMed Central

    Hou, Yong-Feng; Gao, Si-Hai; Wang, Ping; Zhang, He-Mei; Liu, Li-Zhi; Ye, Meng-Xuan; Zhou, Guang-Ming; Zhang, Zeng-Li; Li, Bing-Yan

    2016-01-01

    Ovarian cancer is the most lethal gynecological malignancy due to its high metastatic ability. Epithelial-mesenchymal transition (EMT) is essential during both follicular rupture and epithelium regeneration. However, it may also accelerate the progression of ovarian carcinomas. Experimental studies have found that 1α,25-dihydroxyvitamin-D3 [1α,25(OH)2D3] can inhibit the proliferation of ovarian cancer cells. In this study, we investigated whether 1α,25(OH)2D3 could inhibit the migration of ovarian cancer cells via regulating EMT. We established a model of transient transforming growth factor-β1(TGF-β1)-induced EMT in human ovarian adenocarcinoma cell line SKOV-3 cells. Results showed that, compared with control, 1α,25(OH)2D3 not only inhibited the migration and the invasion of SKOV-3 cells, but also promoted the acquisition of an epithelial phenotype of SKOV-3 cells treated with TGF-β1. We discovered that 1α,25(OH)2D3 increased the expression of epithelial marker E-cadherin and decreased the level of mesenchymal marker, Vimentin, which was associated with the elevated expression of VDR. Moreover, 1α,25(OH)2D3 reduced the expression level of transcription factors of EMT, such as slug, snail, and β-catenin. These results indicate that 1α,25(OH)2D3 suppresses the migration and invasion of ovarian cancer cells by inhibiting EMT, implying that 1α,25(OH)2D3 might be a potential therapeutic agent for the treatment of ovarian cancer. PMID:27548154

  3. 1α,25(OH)2D3-dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells.

    PubMed

    Buitrago, Claudia G; Arango, Nadia S; Boland, Ricardo L

    2012-04-01

    We previously reported that 1α,25-dihydroxy-vitamin D(3) [1α,25(OH)(2)D(3)] induces non-transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)(2)D(3) activates Akt by phosphorylation in Ser473 in a time-dependent manner (5-60 min). When these cells were pretreated with methyl-beta-cyclodextrin to disrupt caveolae microdomains, hormone-induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin-1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)(2)D(3) activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK-activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)(2)D(3) was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone-induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)(2)D(3) also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)(2)D(3). Altogether, these data suggest that 1α,25(OH)(2)D(3) upregulates Akt through Src, PI(3)K, and p38 MAPK to stimulate myogenesis in C2C12 cells.

  4. Fabricating gradient hydrogel scaffolds for 3D cell culture.

    PubMed

    Chatterjee, Kaushik; Young, Marian F; Simon, Carl G

    2011-05-01

    Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.

  5. Exosomal microRNA miR-1246 induces cell motility and invasion through the regulation of DENND2D in oral squamous cell carcinoma

    PubMed Central

    Sakha, Sujata; Muramatsu, Tomoki; Ueda, Koji; Inazawa, Johji

    2016-01-01

    Metastasis is associated with poor prognosis in cancers. Exosomes, which are packed with RNA and proteins and are released in all biological fluids, are emerging as an important mediator of intercellular communication. However, the function of exosomes remains poorly understood in cancer metastasis. Here, we demonstrate that exosomes isolated by size-exclusion chromatography from a highly metastatic human oral cancer cell line, HOC313-LM, induced cell growth through the activation of ERK and AKT as well as promoted cell motility of the poorly metastatic cancer cell line HOC313-P. MicroRNA (miRNA) array analysis identified two oncogenic miRNAs, miR-342–3p and miR-1246, that were highly expressed in exosomes. These miRNAs were transferred to poorly metastatic cells by exosomes, which resulted in increased cell motility and invasive ability. Moreover, miR-1246 increased cell motility by directly targeting DENN/MADD Domain Containing 2D (DENND2D). Taken together, our findings support the metastatic role of exosomes and exosomal miRNAs, which highlights their potential for applications in miRNA-based therapeutics. PMID:27929118

  6. Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction.

    PubMed

    Mathew, Stephen O; Chaudhary, Pankaj; Powers, Sheila B; Vishwanatha, Jamboor K; Mathew, Porunelloor A

    2016-10-18

    Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. Previously, we identified Lectin-like transcript-1 (LLT1/OCIL/CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. In this study, we have found that LLT1 was overexpressed on prostate cancer cell lines (DU145, LNCaP, 22Rv1 and PC3) and in primary prostate cancer tissues both at the mRNA and protein level. We further showed that LLT1 is retained intracellularly in normal prostate cells with minimal cell surface expression. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb on prostate cancer cells increased the NK-mediated cytotoxicity of prostate cancer cells. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.

  7. Enrichment of cancer stem cell-like cells by culture in alginate gel beads.

    PubMed

    Xu, Xiao-xi; Liu, Chang; Liu, Yang; Yang, Li; Li, Nan; Guo, Xin; Sun, Guang-wei; Ma, Xiao-jun

    2014-05-10

    Cancer stem cells (CSCs) are most likely the reason of cancer reoccurrence and metastasis. For further elucidation of the mechanism underlying the characteristics of CSCs, it is necessary to develop efficient culture systems to culture and expand CSCs. In this study, a three-dimensional (3D) culture system based on alginate gel (ALG) beads was reported to enrich CSCs. Two cell lines derived from different histologic origins were encapsulated in ALG beads respectively and the expansion of CSCs was investigated. Compared with two-dimensional (2D) culture, the proportion of cells with CSC-like phenotypes was significantly increased in ALG beads. Expression levels of CSC-related genes were greater in ALG beads than in 2D culture. The increase of CSC proportion after being cultured within ALG beads was further confirmed by enhanced tumorigenicity in vivo. Moreover, increased metastasis ability and higher anti-cancer drug resistance were also observed in 3D-cultured cells. Furthermore, we found that it was hypoxia, through the upregulation of hypoxia-inducible factors (HIFs) that occurred in ALG beads to induce the increasing of CSC proportion. Therefore, ALG bead was an efficient culture system for CSC enrichment, which might provide a useful platform for CSC research and promote the development of new anti-cancer therapies targeting CSCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

    PubMed

    Park, Ji Eun; Park, Min Hee; Kim, Min Seong; Park, Yeo Reum; Yun, Jung Im; Cheong, Hee Tae; Kim, Minseok; Choi, Jung Hoon; Lee, Eunsong; Lee, Seung Tae

    2017-08-17

    Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research. © 2017 International Federation for Cell Biology.

  9. Chelerythrine delayed tumor growth and increased survival duration of Dalton's lymphoma bearing BALB/c H(2d) mice by activation of NK cells in vivo.

    PubMed

    Kumar, Sanjay; Tomar, Munendra Singh; Acharya, Arbind

    2015-01-01

    The aims of the present investigation were to evaluate the antitumor effect of chelerythrine (CHE) on in vivo growth and survival duration of BALB/c (H2d) mice bearing Dalton's lymphoma (DL) and enhanced function of tumor associated NK cells (TANK cells). BALB/c (H2d) mice at 8-10 weeks of age of either sex were used. Increasing concentration of CHE (1.25, 2.5, and 5.0 mg/kg), staurosporine (0.625, 1.0, 1.5, and 2.0 mg/kg) and cyclophosphamide (25, 50, 100, and 200 mg/kg) were administered intraperitoneally and tumor regression and survival duration of tumor bearing host were determined, and thereafter expression of NKG2D and NKG2A on TANK cells were detected. Our results show that treatment with 2.5 mg/kg of CHE results in a significant reduction in mean tumor volume and increased survival duration of DL bearing BALB/c (H2d) mice when compared to control. Activating receptor NKG2D on TANK cells were observed upregulated in contrast to inhibitory receptor NKG2A. CHE reduced mean tumor volume and increased survival duration of DL bearing BALB/c (H2d) mice. Increased expression of activating receptor NKG2D on TANK cells results in recovery of immunosuppression during tumor progression. Therefore, CHE could be a potential anticancer therapeutic agent that may be used to replace chemo-radio-therapy in future.

  10. IDO metabolite produced by EBV-transformed B cells inhibits surface expression of NKG2D in NK cells via the c-Jun N-terminal kinase (JNK) pathway.

    PubMed

    Song, Hyunkeun; Park, Hyunjin; Kim, Jiyoung; Park, Gabin; Kim, Yeong-Seok; Kim, Sung Mok; Kim, Daejin; Seo, Su Kil; Lee, Hyun-Kyung; Cho, DaeHo; Hur, Daeyoung

    2011-05-01

    Natural Killer cells are known to play a major role in the innate immune response against viral infections and tumor cells. Several viruses, such as CMV, EBV and HIV-1, have acquired strategies to escape elimination by NK cells. In this study, we observed that EBV infection increased expression of IDO on B cells. To evaluate the function of IDO associated with EBV infection, we investigated whether EBV-induced IDO could modulate expression of NK cell-activation receptor, NKG2D. When NK cells were co-incubated with EBV transformed B cells, surface expression of NKG2D was significantly reduced in NK cells. Incubation with L-kynurenine, an IDO metabolite, down-modulated NKG2D expression in NK cells in a dose- and time-dependent manner. Incubation with the JNK inhibitor SP600125 also inhibited NKG2D expression in NK cells. In addition, we observed that the effect of L-kynurenine was blocked by JNK agonist, anisomycin, suggesting the involvement of the JNK pathway in the signal transduction of L-kynurenine-reduced NKG2D expression. Furthermore, IL-18 significantly reduced L-kynurenine-induced down-regulation of NKG2D expression in NK cells. Taken together, these data indicate that down-regulation of NKG2D by EBV-induced IDO metabolite provides a potential mechanism by which EBV escapes NKG2D-mediated attack by immune cells.

  11. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    PubMed

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  12. GEL-STATE NMR OF BALL-MILLED WHOLE CELL WALLS IN DMSO-d6 USING 2D SOLUTION-STATE NMR SPECTROSCOPY

    USDA-ARS?s Scientific Manuscript database

    Plant cell walls were used for obtaining 2D solution-state NMR spectra without actual solubilization or structural modification. Ball-milled whole cell walls were swelled directly in the NMR tube with DMSO-d6 where they formed a gel. There are relatively few gel-state NMR studies. Most have involved...

  13. Phenotypic characterization of prostate cancer LNCaP cells cultured within a bioengineered microenvironment.

    PubMed

    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

  14. 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

  15. Downregulation of Ubiquitin-conjugating Enzyme UBE2D3 Promotes Telomere Maintenance and Radioresistance of Eca-109 Human Esophageal Carcinoma Cells

    PubMed Central

    Yang, Hui; Wu, Lin; Ke, Shaobo; Wang, Wenbo; Yang, Lei; Gao, Xiaojia; Fang, Hongyan; Yu, Haijun; Zhong, Yahua; Xie, Conghua; Zhou, Fuxiang; Zhou, Yunfeng

    2016-01-01

    Ubiquitin-conjugating enzyme UBE2D3 is an important member of the ubiquitin-proteasome pathways. Our previous study showed that the expression of UBE2D3 was negatively related to human telomerase reverse transcriptase (hTERT) and radioresistance in human breast cancer cells. However, in esophageal carcinoma, the exact effects and mechanisms of UBE2D3 in radioresistance remain unclear. This study shows that UBE2D3 knockdown was associated with significant increases in radioresistance to X-rays, telomerase activity, telomere length, and telomere shelterins. UBE2D3 knockdown-mediated radioresistance was related to a decrease in the spontaneous and ionizing radiation-induced apoptosis, resulting from a decrease in the Bax/Bcl-2 ratio. Furthermore, UBE2D3 downregulation was associated with increased G1-S phase transition and prolonged IR-induced G2/M arrest through over expression of cyclin D1, decrease of CDC25A expression and promotion of the ATM/ATR-Chk1-CDC25C pathway. Moreover, UBE2D3 downregulation reduced spontaneous DNA double-strand breaks and accelerated the repair of DNA damage induced by IR. The current data thus demonstrate that UBE2D3 downregulation enhances radioresistance by increased telomere homeostasis and prolonged IR-induced G2/M arrest, but decreases the IR-induced apoptosis and the number of DNA damage foci. These results suggest that UBE2D3 might be a potential molecular target to improve radiotherapy effects in esophageal carcinoma. PMID:27326259

  16. Electromagnetic 2D/3D Particle-in-Cell simulations of the solar wind interaction with lunar crustal anomalies.

    NASA Astrophysics Data System (ADS)

    Deca, Jan; Lapenta, Giovanni; Lembège, Bertrand; Divin, Andrey; Markidis, Stefano; Amaya, Jorge

    2013-04-01

    We present the first 2D/3D fully kinetic Particle-in-Cell simulations of the solar wind interaction with lunar crustal magnetic anomalies. The simulations are performed using the implicit electromagnetic Particle-in-Cell code iPIC3D [Markidis, Lapenta & Rizwan-uddin, 2010]. Multiscale physics is resolved for all plasma components (heavy ions, protons and electrons) in the code, recently updated with a set of open boundary conditions designed for solar wind-body interactions. We use a dipole to model the crustal anomaly. The dipole center is located outside the computational domain and the boundary representing the lunar surface is modeled as a particle-absorbing plane. Photo-emission from the lunar surface is at this point not included, but will be in future work. We study the behaviour of the dipole model with variable surface magnetic field strength under changing solar wind conditions and confirm that lunar crustal magnetic fields may indeed be strong enough to stand off the solar wind and form a mini-magnetosphere, as suggested by MHD simulations [Harnett & Winglee, 2000, 2002, 2003] and spacecraft observations [Kurata et al., 2005; Halekas et al., 2008; Wieser et al., 2010]. 3D-PIC simulations reveal to be very helpful to analyze the diversion/braking of the particle flux and the characteristics of the resulting particles accumulation. The particle flux to the surface is significantly reduced at the magnetic anomaly, surrounded by a region of enhanced density due to the magnetic mirror effect. Finally we will present preliminary results on the interaction of the solar wind with weaker magnetic anomalies in which highly non-adiabatic interactions are expected.

  17. The miR-7 Identified from Collagen Biomaterial-Based Three-Dimensional Cultured Cells Regulates Neural Stem Cell Differentiation

    PubMed Central

    Cui, Yi; Xiao, Zhifeng; Chen, Tong; Wei, Jianshu; Chen, Lei; Liu, Lijun; Chen, Bing; Wang, Xiujie; Li, Xiaoran

    2014-01-01

    Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures. PMID:24200387

  18. mTOR inhibition potentiates cytotoxicity of Vγ4 γδ T cells via up-regulating NKG2D and TNF-α.

    PubMed

    Cao, Guangchao; Wang, Qian; Li, Guangqiang; Meng, Ziyu; Liu, Hui; Tong, Jiyu; Huang, Wanjun; Liu, Zonghua; Jia, Yanqiong; Wei, Jun; Chi, Hongbo; Yang, Hengwen; Zhao, Liqing; Wu, Zhenzhou; Hao, Jianlei; Yin, Zhinan

    2016-11-01

    γδ T cells play a critical role in early anti-tumor immunity and perform cytotoxicity via NKG2D for recognition and multiple cytotoxic factors for tumor killing. Recent studies have demonstrated pivotal roles of mTOR-mediated metabolism in the maturation, differentiation, and effector function of diverse immune cells, including DCs, NK cells, CD4(+) T cell subsets, and CD8(+) T cells, but the role of mTOR signaling in γδ T cells is barely known. Here, we showed that suppressing mTOR signaling in in vitro-expanded Vγ4 γδ T cells via the mechanistic inhibitor rapamycin enhanced their cytotoxicity against multiple tumor cell lines, and these cells performed better tumor-suppressing effects upon adoptive therapy. Further investigation revealed that elevated cytotoxicity was a result of up-regulation of NKG2D and TNF-α. Moreover, rapamycin treatment significantly decreased the expression of CISH and increased pSTAT5. The inhibition of STAT5 pathways via siRNA interference or a specific inhibitor eliminated the up-regulation of NKG2D and TNF-α in rapamycin-treated Vγ4 γδ T cells. These results uncovered an important role of mTOR signaling in the cytotoxic effector function of γδ T cells and provided a potential strategy to improve γδ T cell-based cancer immunotherapy. © Society for Leukocyte Biology.

  19. Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells.

    PubMed

    Wan, Carthur K; O'Carroll, Simon J; Kim, Sue-Ling; Green, Colin R; Nicholson, Louise F B

    2013-12-01

    While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models. A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01). Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

  20. Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells.

    PubMed

    Zhu, Li; Tatsuke, Tsuneyuki; Xu, Jian; Li, Zhiqing; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2015-09-01

    The phenomenon of RNA interference (RNAi) has been found in various organisms. However, the proteins implicated in RNAi pathway in different species show distinct roles. Knowledge on the underlying mechanism of lepidopteron RNAi is quite lacking such as the roles of Loquacious (Loqs) and R2D2, the dsRNA-binding proteins in silkworm RNAi pathway. Here, we report that Loqs and R2D2 protein depletion affected efficiency of dsRNA-mediated RNAi pathway. Besides, Loqs was found to co-localize with Dicer2 to some specific cytoplasmic foci, which were looked like D2-bodies marked by R2D2 and Dicer2 in Fly cells, thereby calling the foci as D2 body-like granules. Using RNAi methods, Loqs was found to be the key protein in these granules, although R2D2 determined the localization of Loqs in D2 body-like granules. Interestingly, in the R2D2-depeted silkworm cells, the formation of processing bodies, another cytoplasmic foci, was affected. These data indicated R2D2 regulated these two kinds of cytoplasmic foci. Domain deletion analysis demonstrated that dsRBD 1 and 2 were required for Loqs in D2 body-like granules and dsRBD 2 and 3 were required for Loqs to interact with R2D2 and Ago1, respectively. Altogether, our observations provide important information for further study on D2 body-like granules, the newly found cytoplasmic foci in silkworm cells.

  1. Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms

    PubMed Central

    Kloxin, April M.; Tibbitt, Mark W.; Anseth, Kristi S.

    2013-01-01

    We describe a detailed procedure to create photolabile, poly(ethylene glycol)-based (PEG) hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in 2D or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. Here, we provide coupling procedures to PEG-bis-amine to form a photodegradable crosslinker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. Hydrogels are synthesized with the photodegradable crosslinker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. Cell-material interactions can be probed in 2D or 3D by spatiotemporally controlling the gel microenvironment, which allows unique experiments to be performed to monitor cell response to changes in their niche. Degradation is readily achieved with cytocompatible wavelengths of low intensity flood irradiation (365 to 420 nm) in minutes or with highintensity laser irradiation (405 nm) in seconds. In this protocol, synthesis and purification of the photodegradable monomers take approximately 2 weeks, but can be substantially shortened by purchasing the o-nitrobenzylether precursor. Preparation of the sterile solutions for hydrogel fabrication takes hours, while the reaction to form the final hydrogel is complete in minutes. Hydrogel degradation occurs on-demand, in seconds to minutes, with user-directed light exposure. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. These PEG-based materials are useful for probing the dynamic influence of cell-cell and cell-material interactions on cell function in 2D or 3D. While other protocols are available for controlling peptide presentation or modulus, few allow manipulation of material properties in situ and in the

  2. [Non-small cell lung cancer 95D cells co-cultured with 3D-bioprinted scaffold to construct a lung cancer model in vitro].

    PubMed

    Mou, Hao; Wang, Jian; Hu, Huizhen; Xu, Wei; Chen, Qingyong

    2015-10-01

    To fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions. We chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis. Cells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05). The cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold

  3. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  4. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  5. 1,25(OH)2D3 induces regulatory T cell differentiation by influencing the VDR/PLC-γ1/TGF-β1/pathway.

    PubMed

    Zhou, Qiang; Qin, Shengying; Zhang, Jinyan; Zhon, Lin; Pen, Zhihai; Xing, Tonghai

    2017-09-16

    Vitamin D has been recommended as an immune modulator in recent years, in addition to regulating calcium-phosphorous-bone metabolism. Clinical studies on organ transplantation found that vitamin D sufficiency patients were less likely to develop acute cellular rejection within one year after transplantation compared to those with vitamin D deficiency. Thus, a high percentage of regulatory T cells might play a key role in preventing acute cellular rejection (ACR). In this report, we studied the specific effects of 1,25(OH)2D3 on human T cell diff ;erentiation, and determined the potential molecule mechanism behind. Results showed that 1,25(OH)2D3 induced the differentiation of T-regulatory cells (Treg cells), while inhibiting Th17 cell proliferation. In addition, 1,25(OH)2D3 promoted secretion of the anti-inflammatory cytokine, transforming Growth Factor beta1 (TGF-β1) but suppressed pro-inflammatory cytokines such as interleukin-17 (IL-17). Phospholipase C gamma 1 (PLC-γ1) is an indispensable signaling protein downstream of the classical TCR signaling pathway and was shown to play a crucial role in T cell activation, while Naive T cells expressed less PLC-γ1. Here we showed that Vitamin D could significantly upregulate PLC-γ1 expression, which then induced expression of TGF-β1. In summary, 1,25(OH)2D3 indirectly modulates the differentiation of Treg/Th17 cells by aff ;ecting the VDR/PLC-γ1/TGF-β1pathway. These results indicate that administration 1,25(OH)2D3 supplements may be a beneficial treatment for organ transplantation recipients. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Membrane actions of 1α,25(OH)2D3 are mediated by Ca(2+)/calmodulin-dependent protein kinase II in bone and cartilage cells.

    PubMed

    Doroudi, Maryam; Plaisance, Marc C; Boyan, Barbara D; Schwartz, Zvi

    2015-01-01

    1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Cell sources for in vitro human liver cell culture models

    PubMed Central

    Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-01-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

  8. Cell sources for in vitro human liver cell culture models.

    PubMed

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

  9. 1,25(OH)2D3 disrupts glucose metabolism in prostate cancer cells leading to a truncation of the TCA cycle and inhibition of TXNIP expression.

    PubMed

    Abu El Maaty, Mohamed A; Alborzinia, Hamed; Khan, Shehryar J; Büttner, Michael; Wölfl, Stefan

    2017-10-01

    Prostate cell metabolism exhibits distinct profiles pre- and post-malignancy. The malignant metabolic shift converts prostate cells from "citrate-producing" to "citrate-oxidizing" cells, thereby enhancing glucose metabolism, a phenotype that contrasts classical tumoral Warburg metabolism. An on-line biosensor chip system (BIONAS 2500) was used to monitor metabolic changes (glycolysis and respiration) in response to the putative anti-cancer nutraceutical 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in different prostate cancer (PCa) cell lines (LNCaP, VCaP, DU145 and PC3). LNCaP cells exhibited profound metabolic responsiveness to the treatment and thus extensive analysis of metabolism-modulating effects of 1,25(OH)2D3 were performed, including mRNA expression analysis of key metabolic genes (e.g. GLUT1 and PDHK1), analysis of TCA cycle metabolites, glucose uptake/consumption measurements, ATP production, and mitochondrial biogenesis/activity. Altogether, data demonstrate a vivid disruption of glucose metabolism by 1,25(OH)2D3, illustrated by a decreased glucose uptake and an accumulation of citrate/isocitrate due to TCA cycle truncation. Depletion of glycolytic intermediates led to a consistent decrease in TXNIP expression in response to 1,25(OH)2D3, an effect that coincided with the activation of AMPK signaling and a reduction in c-MYC expression. Reduction in TXNIP levels in response to 1,25(OH)2D3 was rescued by an AMPK signaling inhibitor and mimicked by a MYC inhibitor highlighting the possible involvement of both pathways in mediating 1,25(OH)2D3's metabolic effects in PCa cells. Furthermore, pharmacological and genetic modulation of the androgen receptor showed similar and disparate effects on metabolic parameters compared to 1,25(OH)2D3 treatment, highlighting the AR-independent nature of 1,25(OH)2D3's metabolism-modulating effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. The inhibitory effects of synthetic short peptides, mimicking MICA and targeting at NKG2D receptors, on function of NK cells.

    PubMed

    Zhang, Bin; Wei, Haiming; Zheng, Xiaodong; Zhang, Jian; Sun, Rui; Tian, Zhigang

    2005-03-01

    NKG2D is an activating receptor expressed on most of human NK cells, one of whose ligands is MICA. Based on the crystal structure of NKG2D-MICA complex, we synthesized three short peptides (P1, P2 and P3), mimicking functional alpha1 and alpha2 domain of MICA. The inhibitory effects of three peptides on NK-92 cells, a human NK cell line against Hela cells were observed and the inhibitory percentage was 38% at maximum for P1+P2+P3 in concentration of 1nM. The same peptides had no effect on NK-92 cell against target cells lacking MICA (K562 cells line). The unrelated peptides as controls had no effect on the system. Two peptides (P2 and P3) were prolonged at one or both ends, and the longer forms of peptides exerted stronger inhibitory effects than their shorter forms. Each combination of two peptides exerted a stronger function than single peptide (P1, P2, P3), indicating that shedding of longer amino acid sequence of alpha1 domain or more domain sites of MICA are better than shorter sequence and fewer sites. P1+P2+P3 revealed the almost same inhibitory rate as the soluble MICA (sMICA). P1+P2+P3 were also able to alleviate the concanavalin A-induced murine autoimmune hepatitis in vivo, conforming the similarity of NKG2D between human and mice. The results demonstrate that MICA-mimicking peptides will be useful to search the specific functional sites for NKG2D-MICA interaction, but also promising in explaining NKG2D-related autoimmunity.

  11. Modulation of NKG2D-mediated cytotoxic functions of natural killer cells by viral protein R from HIV-1 primary isolates.

    PubMed

    Pham, Tram N Q; Richard, Jonathan; Gerard, Francine C A; Power, Christopher; Cohen, Éric A

    2011-12-01

    HIV-1 viral protein R (Vpr) from laboratory-adapted virus strains activates the DNA damage/stress sensor ATR kinase and induces cell cycle arrest at the G(2)/M phase through a process that requires Vpr to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex. Activation of this DNA damage/stress checkpoint in G(2) by Vpr was shown to modulate NKG2D-dependent NK cell effector functions via enhancing expression of NKG2D ligands, notably ULBP2. However, it is unknown whether Vpr from HIV-1 primary isolates (groups M, N, O, and P) could modulate NKG2D-mediated cytotoxic functions of NK cells. Here, we report that Vpr from most HIV-1 primary isolates can upregulate ULBP2 expression and induce NKG2D-dependent NK cell killing. Importantly, these activities were always accompanied by an active G(2) cell cycle arrest function. Interestingly, Vpr variants from group P and a clade D isolate of group M were defective at enhancing NKG2D-mediated NK cell lysis owing to their inability to augment ULBP2 expression. However, distinct mechanisms were responsible for their failure to do so. While Vpr from group P was deficient in its ability to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex, the Vpr variant from group D was unable to properly localize to the nucleus, underlining the importance of these biological properties in Vpr function. In conclusion, the ability of Vpr from HIV-1 primary isolates to regulate NK cell effector function underscores the importance of this HIV-1 accessory protein in the modulation of the host's innate immune responses.

  12. A microfluidic system for automatic cell culture

    NASA Astrophysics Data System (ADS)

    Huang, Chun-Wei; Lee, Gwo-Bin

    2007-07-01

    This study presents a new chip capable of automating the cell culture process by using microfluidic technology. This microfluidic cell culture system comprising microheaters, a micro temperature sensor, micropumps, microvalves, microchannels, a cell culture area and several reservoirs was fabricated by using micro-electro-mechanical-systems' fabrication processes. Traditional manual cell culture processes can be performed on this chip. A uni-directional pneumatic micropump was developed to transport the culture reagents and constraint the solutions to flow only in one direction, safeguarding the entire culture process from contamination. A new micro check valve was also used to prevent the culture solutions from flowing back into the microchannels. The microheaters and the micro temperature sensor were used to maintain a constant temperature during the cell culturing process. The pH value suitable for cell growth was also regulated during the cell culture process. A typical cell culturing process for human lung cancer cells (A549) was successfully performed to demonstrate the capability of the developed microfluidic system. This automatic cell culturing system can be eventually integrated with subsequent microfluidic modules for cell purification, collection, counting and lysis to form a cell-based micro-total-analysis system. Preliminary results have been presented in The Asia-Pacific Conference of Transducers and Micro-Nano Technology (APCOT), 25-28 June 2006

  13. Cell cycle arrest and apoptosis induced by 1α,25(OH)2D3 and TX 527 in Kaposi sarcoma is VDR dependent.

    PubMed

    González-Pardo, Verónica; Suares, Alejandra; Verstuyf, Annemieke; De Clercq, Pierre; Boland, Ricardo; de Boland, Ana Russo

    2014-10-01

    We have previously shown that 1α,25(OH)2-Vitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 inhibit the proliferation of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR) and this could be partially explained by the inhibition of the NF-κB pathway. In this work, we further explored the mechanism of action of both vitamin D compounds in Kaposi sarcoma. We investigated whether the cell cycle arrest and subsequent apoptosis of endothelial cells (SVEC) and SVEC transformed by vGPCR (SVEC-vGPCR) elicited by 1α,25(OH)2D3 and TX 527 were mediated by the vitamin D receptor (VDR). Cell cycle analysis of SVEC and SVEC-vGPCR treated with 1α,25(OH)2D3 (10nM, 48h) revealed that 1α,25(OH)2D3 increased the percentage of cells in the G0/G1 phase and diminished the percentage of cells in the S phase of the cell cycle. Moreover, the number of cells in the S phase was higher in SVEC-vGPCR than in SVEC due to vGPCR expression. TX 527 exerted similar effects on growth arrest in SVEC-vGPCR cells. The cell cycle changes were suppressed when the expression of the VDR was blocked by a stable transfection of shRNA against VDR. Annexin V-PI staining demonstrated apoptosis in both SVEC and SVEC-vGPCR after 1α,25(OH)2D3 and TX 527 treatment (10nM, 24h). Cleavage of caspase-3 detected by Western blot analysis was increased to a greater extent in SVEC than in SVEC-vGPCR cells, and this effect was also blocked in VDR knockdown cells. Altogether, these results suggest that 1α,25(OH)2D3 and TX 527 inhibit the proliferation of SVEC and SVEC-vGPCR and induce apoptosis by a mechanism that involves the VDR.

  14. Basic techniques in mammalian cell tissue culture.

    PubMed

    Phelan, Katy; May, Kristin M

    2015-03-02

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.

  15. A rapid and efficient 2D/3D nuclear segmentation method for analysis of early mouse embryo and stem cell image data.

    PubMed

    Lou, Xinghua; Kang, Minjung; Xenopoulos, Panagiotis; Muñoz-Descalzo, Silvia; Hadjantonakis, Anna-Katerina

    2014-03-11

    Segmentation is a fundamental problem that dominates the success of microscopic image analysis. In almost 25 years of cell detection software development, there is still no single piece of commercial software that works well in practice when applied to early mouse embryo or stem cell image data. To address this need, we developed MINS (modular interactive nuclear segmentation) as a MATLAB/C++-based segmentation tool tailored for counting cells and fluorescent intensity measurements of 2D and 3D image data. Our aim was to develop a tool that is accurate and efficient yet straightforward and user friendly. The MINS pipeline comprises three major cascaded modules: detection, segmentation, and cell position classification. An extensive evaluation of MINS on both 2D and 3D images, and comparison to related tools, reveals improvements in segmentation accuracy and usability. Thus, its accuracy and ease of use will allow MINS to be implemented for routine single-cell-level image analyses.

  16. NKG2D - RAE-1 receptor-ligand variation does not account for the Natural Killer cell defect in nonobese diabetic mice1

    PubMed Central

    Maier, Lisa M.; Howlett, Sarah; Rainbow, Kara; Clark, Jan; Howson, Joanna M.M.; Todd, John A.; Wicker, Linda S.

    2008-01-01

    Natural Killer (NK) cells from nonobese diabetic (NOD) mice induced with poly I:C in vivo exhibit low cytotoxicity against a range of target cells, but the genetic mechanisms controlling this defect are yet to be elucidated. Defects in the expression of NKG2D and its ligands, the RAE-1 molecules, have been hypothesized to contribute to the reduced NK function present in NOD mice. Here we show that segregation of the NK-mediated killing phenotype did not correlate with the NOD Raet1 haplotype, and that the large alterations in NKG2D expression previously reported on NK cells expanded in vitro were not observed in primary, poly I:C-elicited NK cells in vivo. Additional studies indicate a complex genetic control of defective NOD NK cells, including genes linked to the MHC and possibly those that are associated with an altered cytokine response to the TLR3-agonist, poly I:C. PMID:18981127

  17. 1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells.

    PubMed

    Zeng, Ni; Zhou, Yuetao; Zhang, Shaqiu; Singh, Yogesh; Shi, Bing; Salker, Madhuri S; Lang, Florian

    2017-01-01

    Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions and lactate, which is in part accomplished by Na+/H+ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)2D3 on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)2D3 on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96®, Annexin V and Propidium Iodide staining. A 24 hours treatment with 1,25(OH)2D3 (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na+/H+ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)2D3 (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. In Ishikawa cells 1,25(OH)2D3 is a powerful stimulator of glycolysis, an effect presumably due to

  18. Dynamized Preparations in Cell Culture

    PubMed Central

    Sunila, Ellanzhiyil Surendran; Preethi, Korengath Chandran; Kuttan, Girija

    2009-01-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  19. Computer-aided 2D and 3D quantification of human stem cell fate from in vitro samples using Volocity high performance image analysis software.

    PubMed

    Piltti, Katja M; Haus, Daniel L; Do, Eileen; Perez, Harvey; Anderson, A J; Cummings, B J

    2011-11-01

    Accurate automated cell fate analysis of immunostained human stem cells from 2- and 3-dimensional (2D-3D) images would improve efficiency in the field of stem cell research. Development of an accurate and precise tool that reduces variability and the time needed for human stem cell fate analysis will improve productivity and interpretability of the data across research groups. In this study, we have created protocols for high performance image analysis software Volocity® to classify and quantify cytoplasmic and nuclear cell fate markers from 2D-3D images of human neural stem cells after in vitro differentiation. To enhance 3D image capture efficiency, we optimized the image acquisition settings of an Olympus FV10i® confocal laser scanning microscope to match our quantification protocols and improve cell fate classification. The methods developed in this study will allow for a more time efficient and accurate software based, operator validated, stem cell fate classification and quantification from 2D and 3D images, and yield the highest ≥94.4% correspondence with human recognized objects.

  20. 25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3

    PubMed Central

    Chiang, Kun-Chun; Yeh, Chun-Nan; Huang, Cheng-Cheng; Yeh, Ta-Sen; S. Pang, Jong-Hwei; Hsu, Jun-Te; Chen, Li-Wei; Kuo, Sheng-Fong; Kittaka, Atsushi; Chen, Tai C.; Juang, Horng-Heng

    2016-01-01

    Cholangiocarcinoma (CCA) is a devastating disease without effective treatments. 1α,25(OH)2D3, the active form of Vitamin D, has emerged as a new anti-cancer regimen. However, the side effect of hypercalcemia impedes its systemic administration. 25(OH)D is biologically inert and needs hydroxylation by CYP27B1 to form 1α,25(OH)2D3, which is originally believed to only take place in kidneys. Recently, the extra-renal expression of CYP27B1 has been identified and in vitro conversion of 25(OH)D to 1α,25(OH)2D3 has been found in some cancer cells with CYP27B1 expression. In this study, CYP27B1 expression was demonstrated in CCA cells and human CCA specimens. 25(OH)D effectively represses SNU308 cells growth, which was strengthened or attenuated as CYP27B1 overexpression or knockdown. Lipocalcin-2 (LCN2) was also found to be repressed by 25(OH)D. After treatment with 800 ng/mL 25(OH)D, the intracellular 1α,25(OH)2D3 concentration was higher in SNU308 cells with CYP27B1 overexpression than wild type SNU308 cells. In a xenograft animal experiment, 25(OH)D, at a dose of 6 μg/kg or 20 μg/kg, significantly inhibited SNU308 cells’ growth without inducing obvious side effects. Collectively, our results indicated that SNU308 cells were able to convert 25(OH)D to 1α,25(OH)2D3 and 25(OH)D CYP27B1 gene therapy could be deemed as a promising therapeutic direction for CCA. PMID:27529229

  1. Histone deacetylase inhibitors enhance expression of NKG2D ligands in Ewing sarcoma and sensitize for natural killer cell-mediated cytolysis

    PubMed Central

    2012-01-01

    Background Ewing sarcoma patients have a poor prognosis despite multimodal therapy. Integration of combination immunotherapeutic strategies into first-/second-line regimens represents promising treatment options, particularly for patients with intrinsic or acquired resistance to conventional therapies. We evaluated the susceptibility of Ewing sarcoma to natural killer cell-based combination immunotherapy, by assessing the capacity of histone deacetylase inhibitors to improve immune recognition and sensitize for natural killer cell cytotoxicity. Methods Using flow cytometry, ELISA and immunohistochemistry, expression of natural killer cell receptor ligands was assessed in chemotherapy-sensitive/-resistant Ewing sarcoma cell lines, plasma and tumours. Natural killer cell cytotoxicity was evaluated in Chromium release assays. Using ATM/ATR inhibitor caffeine, the contribution of the DNA damage response pathway to histone deacetylase inhibitor-induced ligand expression was assessed. Results Despite comparable expression of natural killer cell receptor ligands, chemotherapy-resistant Ewing sarcoma exhibited reduced susceptibility to resting natural killer cells. Interleukin-15-activation of natural killer cells overcame this reduced sensitivity. Histone deacetylase inhibitor-pretreatment induced NKG2D-ligand expression in an ATM/ATR-dependent manner and sensitized for NKG2D-dependent cytotoxicity (2/4 cell lines). NKG2D-ligands were expressed in vivo, regardless of chemotherapy-response and disease stage. Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls. Conclusion Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma. PMID:22587892

  2. Microfluidic 3D cell culture: potential application for tissue-based bioassays

    PubMed Central

    Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

    2014-01-01

    Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

  3. Valproic Acid Upregulates NKG2D Ligand Expression through an ERK-dependent Mechanism and Potentially Enhances NK Cell-mediated Lysis of Myeloma1

    PubMed Central

    Wu, Xiaosong; Tao, Yi; Hou, Jun; Meng, Xiuqin; Shi, Jumei

    2012-01-01

    Modulation of the antitumor immune response through the engagement of NKG2D receptors with their ligands (L) on targets represents a promising therapeutic approach against cancer. In this study, we tested the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the expression of NKG2D ligands in myeloma cells. We demonstrated that VPA was able to upregulate both protein and mRNA expression of major histocompatibility complex class I-related chain (MIC) A/B and UL16-binding protein (ULBP) 2 without any significant effect on the expression of ULBP1, ULBP3, and ULBP4 or induction of other natural killer (NK) cell ligands, such as NKp30-L, NKp44-L, and NKp46-L in myeloma cells. A 51Cr release assay and degranulation assay indicated that the induction of MICA/B and ULBP2 augmented NK cell-mediated lysis of myeloma cells, which was abolished by the addition of a blocking NKG2D antibody. Activation of constitutively phosphorylated extracellular signal-regulated kinase (ERK) by VPA is essential for the up-regulation of MICA/B and ULBP2 expressions. Inhibition of ERK using ERK inhibitor PD98059 decreased both MICA/B and ULBP2 expressions and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in ARK resulted in increased MICA/B and ULBP2 expressions and enhanced NK cell lysis. These data indicate that increased sensitivity of VPA-treated myeloma cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. Our results provide evidence that targeting ERK signaling pathway may be an additional mechanism supporting the antimyeloma activity of HDAC inhibitors and suggest its possible immunotherapeutic value for myeloma treatment. PMID:23308050

  4. Dacarbazine-mediated upregulation of NKG2D ligands on tumor cells activates NK and CD8 T cells and restrains melanoma growth.

    PubMed

    Hervieu, Alice; Rébé, Cédric; Végran, Frédérique; Chalmin, Fanny; Bruchard, Mélanie; Vabres, Pierre; Apetoh, Lionel; Ghiringhelli, François; Mignot, Grégoire

    2013-02-01

    Dacarbazine (DTIC) is a cytotoxic drug widely used for melanoma treatment. However, the putative contribution of anticancer immune responses in the efficacy of DTIC has not been evaluated. By testing how DTIC affects host immune responses to cancer in a mouse model of melanoma, we unexpectedly found that both natural killer (NK) and CD8(+) T cells were indispensable for DTIC therapeutic effect. Although DTIC did not directly affect immune cells, it triggered the upregulation of NKG2D ligands on tumor cells, leading to NK cell activation and IFNγ secretion in mice and humans. NK cell-derived IFNγ subsequently favored upregulation of major histocompatibility complex class I molecules on tumor cells, rendering them sensitive to cytotoxic CD8(+) T cells. Accordingly, DTIC markedly enhanced cytotoxic T lymphocyte antigen 4 inhibition efficacy in vivo in an NK-dependent manner. These results underscore the immunogenic properties of DTIC and provide a rationale to combine DTIC with immunotherapeutic agents that relieve immunosuppression in vivo.

  5. CD155 on HIV-Infected Cells Is Not Modulated by HIV-1 Vpu and Nef but Synergizes with NKG2D Ligands to Trigger NK Cell Lysis of Autologous Primary HIV-Infected Cells.

    PubMed

    Davis, Zachary B; Sowrirajan, Bharatwaj; Cogswell, Andrew; Ward, Jeffery P; Planelles, Vicente; Barker, Edward

    2017-02-01

    Activation of primary CD4(+) T cells induces the CD155, but not the CD112 ligands for the natural killer (NK) cell activation receptor (aNKR) CD226 [DNAX accessory molecule-1 (DNAM-1)]. We hypothesize that HIV productively infects activated CD4(+) T cells and makes itself vulnerable to NK cell-mediated lysis when CD155 on infected T cells engages DNAM-1. The primary objective of this study is to determine whether CD155 alone or together with NKG2D ligands triggers autologous NK cell lysis of HIV-infected T cells and whether HIV modulates CD155. To determine whether HIV modulates this activation ligand, we infected "activated" CD4(+) T cells with HIV in the absence or presence of Nef and/or Vpu and determined by flow cytometry whether they modulated CD155. To determine if CD155 alone, or together with NKG2D ligands, triggered NK cell lysis of autologous HIV-infected T cells, we treated purified NK cells with DNAM-1 and/or NKG2D blocking antibodies before the addition of purified autologous HIV-infected cells in cytolytic assays. Finally, we determined whether DNAM-1 works together with NKG2D as an NK cell coactivation receptor (caNKR) or whether they work independently as aNKRs to induce an NK cell lytic response. We demonstrate that HIV and specifically Nef and/or Vpu do not modulate CD155 on infected primary T cells; and both CD155 and NKG2D ligands synergize as aNKRs to trigger NK cell lysis of the infected cell.

  6. Best practices in cell culture: an overview.

    PubMed

    Baust, John M; Buehring, Gertrude Case; Campbell, Lia; Elmore, Eugene; Harbell, John W; Nims, Raymond W; Price, Paul; Reid, Yvonne A; Simione, Frank

    2017-08-14

    This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

  7. Effects of 1α,25-(OH)2D3 on the formation and activity of osteoclasts in RAW264.7 cells.

    PubMed

    Gu, Jianhong; Tong, Xi-Shuai; Chen, Guo-Hong; Wang, Dong; Chen, Yang; Yuan, Yan; Liu, Xue-Zhong; Bian, Jian-Chun; Liu, Zong-Ping

    2015-08-01

    The hormonally active form of vitamin D3, 1α,25-(OH)2D3, has an important role in bone metabolism. This study examined the effects of 1α,25-(OH)2D3 on the ability of two cytokines, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1α,25-(OH)2D3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin β3, V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATc1 also increased 24h after treatment with 1α,25-(OH)2D3. These results suggest that 1α,25-(OH)2D3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

    PubMed Central

    Okita, Riki; Wolf, Diana; Yasuda, Koichiro; Maeda, Ai; Yukawa, Takuro; Saisho, Shinsuke; Shimizu, Katsuhiko; Yamaguchi, Yoshiyuki; Oka, Mikio; Nakayama, Eiichi; Lundqvist, Andreas; Kiessling, Rolf; Seliger, Barbara; Nakata, Masao

    2015-01-01

    Introduction Several cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown. Methods This study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis. Results We demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a. Conclusion In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells. PMID:26439264

  9. Increased sMICA and TGFβ1 levels in HNSCC patients impair NKG2D-dependent functionality of activated NK cells

    PubMed Central

    Klöß, Stephan; Chambron, Nicole; Gardlowski, Tanja; Arseniev, Lubomir; Koch, Joachim; Esser, Ruth; Glienke, Wolfgang; Seitz, Oliver; Köhl, Ulrike

    2015-01-01

    Disseminated head-and-neck squamous cell carcinoma (HNSCC) escapes immune surveillance and thus frequently manifests as fatal disease. Here, we report on the distribution of distinct immune cell subpopulations, natural killer (NK) cell cytotoxicity and tumor immune escape mechanisms (TIEMs) in 55 HNSCC patients, either at initial diagnosis or present with tumor relapse. Compared to healthy controls, the regulatory NK cells and the ratio of pro/anti-inflammatory cytokines were decreased in HNSCC patients, while soluble major histocompatibility complex Class I chain-related peptide A (sMICA) and transforming growth factor β1 (TGFβ1) plasma levels were markedly elevated. Increased sMICA and TGFβ1 concentrations correlated with tumor progression and staging characteristics in 7 follow-up HNSCC patients, with significantly elevated levels of both soluble factors from the time of initial diagnosis to that of relapse. Patient plasma containing elevated sMICA and TGFβ1 markedly impaired NKG2D-dependent cytotoxicity against HNSCC cells upon incubation with patient-derived and IL-2 activated NK cells vs. those derived from healthy donors. Decreased antitumor recognition was accompanied by reduced NKG2D expression on the NK cell surface and an enhanced caspase-3 activity. In-vitro blocking and neutralization experiments demonstrated a synergistic negative impact of sMICA and TGFβ1 on NK cell functionality. Although we previously showed the feasibility and safety of transfer of allogeneic donor NK cells in a prior clinical study encompassing various leukemia and tumor patients, our present results suggest the need for caution regarding the sole use of adoptive NK cell transfer. The presence of soluble NKG2D ligands in the plasma of HNSCC patients and the decreased NK cell cytotoxicity due to several factors, especially TGFβ1, indicates timely depletion of these immunosuppressing molecules may promote NK cell-based immunotherapy. PMID:26451327

  10. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone) Scaffolds.

    PubMed

    Palomeras, Sònia; Rabionet, Marc; Ferrer, Inés; Sarrats, Ariadna; Garcia-Romeu, Maria Luisa; Puig, Teresa; Ciurana, Joaquim

    2016-04-23

    The cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs' phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone) (PCL), a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.

  11. Augmented anti-tumor activity of NK-92 cells expressing chimeric receptors of TGF-βR II and NKG2D.

    PubMed

    Wang, Zhongjuan; Guo, Linghua; Song, Yuan; Zhang, Yinsheng; Lin, Dandan; Hu, Bo; Mei, Yu; Sandikin, Dedy; Liu, Haiyan

    2017-04-01

    The capacity of natural killer (NK) cells to kill tumor cells without specific antigen recognition provides an advantage over T cells and makes them potential effectors for tumor immunotherapy. However, the efficacy of NK cell adoptive therapy can be limited by the immunosuppressive tumor microenvironment. Transforming growth factor-β (TGF-β) is a potent immunosuppressive cytokine that can suppress NK cell function. To convert the suppressive signal induced by TGF-β to an activating signal, we genetically modified NK-92 cells to express a chimeric receptor with TGF-β type II receptor extracellular and transmembrane domains and the intracellular domain of NK cell-activating receptor NKG2D (TN chimeric receptor). NK-92 cells expressing TN receptors were resistant to TGF-β-induced suppressive signaling and did not down-regulate NKG2D. These modified NK-92 cells had higher killing capacity and interferon γ (IFN-γ) production against tumor cells compared with the control cells and their cytotoxicity could be further enhanced by TGF-β. More interestingly, the NK-92 cells expressing TN receptors were better chemo-attracted to the tumor cells expressing TGF-β. The presence of these modified NK-92 cells significantly inhibited the differentiation of human naïve CD4(+) T cells to regulatory T cells. NK-92-TN cells could also inhibit tumor growth in vivo in a hepatocellular carcinoma xenograft tumor model. Therefore, TN chimeric receptors can be a novel strategy to augment anti-tumor efficacy in NK cell adoptive therapy.

  12. Cytomegalovirus-Associated CD4(+) CD28(null) Cells in NKG2D-Dependent Glomerular Endothelial Injury and Kidney Allograft Dysfunction.

    PubMed

    Shabir, S; Smith, H; Kaul, B; Pachnio, A; Jham, S; Kuravi, S; Ball, S; Chand, S; Moss, P; Harper, L; Borrows, R

    2016-04-01

    Emerging data suggest that expansion of a circulating population of atypical, cytotoxic CD4(+) T cells lacking costimulatory CD28 (CD4(+) CD28(null) cells) is associated with latent cytomegalovirus (CMV) infection. The purpose of the current study was to increase the understanding of the relevance of these cells in 100 unselected kidney transplant recipients followed prospectively for a median of 54 months. Multicolor flow cytometry of peripheral blood mononuclear cells before transplantation and serially posttransplantation was undertaken. CD4(+) CD28(null) cells were found predominantly in CMV-seropositive patients and expanded in the posttransplantation period. These cells were predominantly effector-memory phenotype and expressed markers of endothelial homing (CX3CR1) and cytotoxicity (NKG2D and perforin). Isolated CD4(+) CD27(-) CD28(null) cells proliferated in response to peripheral blood mononuclear cells previously exposed to CMV-derived (but not HLA-derived) antigens and following such priming incubation with glomerular endothelium resulted in signs of endothelial damage and apoptosis (release of fractalkine and von Willebrand factor; increased caspase 3 expression). This effect was mitigated by NKG2D-blocking antibody. Increased CD4(+) CD28(null) cell frequencies were associated with delayed graft function and lower estimated glomerular filtration rate at end follow-up. This study suggests an important role for this atypical cytotoxic CD4(+) CD28(null) cell subset in kidney transplantation and points to strategies that may minimize the impact on clinical outcomes.

  13. Cell Culture as an Alternative in Education.

    ERIC Educational Resources Information Center

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  14. Cell Culture as an Alternative in Education.

    ERIC Educational Resources Information Center

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  15. Cell culture techniques in honey bee research

    USDA-ARS?s Scientific Manuscript database

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  16. Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells

    PubMed Central

    Pan, Dong; Chen, Yaxiong; Du, Yarong; Ren, Zhenxin; Li, Xiaoman; Hu, Burong

    2017-01-01

    Three dimensional (3D) culture in vitro is a new cell culture model that more closely mimics the physiology features of the in vivo environment and is being used widely in the field of medical and biological research. It has been demonstrated that cancer cells cultured in 3D matrices are more radioresistant compared with cells in monolayer (2D). However, the mechanisms causing this difference remain largely unclear. Here we found that the cell cycle distribution and expression of cell cycle regulation genes in 3D A549 cells are different from the 2D. The higher levels of the promotor methylation of cell cycle regulation genes such as RBL1 were observed in 3D A549 cells compared with cells in 2D. The treatments of irradiation or 5-Aza-CdR activated the demethylation of RBL1 promotor and resulted in the increased expression of RBL1 only in 3D A549 cells. Inhibition of RBL1 enhanced the radioresistance and decreased the G2/M phase arrest induced by irradiation in 2D A549 and MCF7 cells. Overexpression of RBL1 sensitized 3D cultured A549 and MCF7 cells to irradiation. Taken together, to our knowledge, it is the first time to revealthat the low expression of RBL1 due to itself promotor methylation in 3D cells enhances the radioresistance. Our finding sheds a new light on understanding the features of the 3D cultured cell model and its application in basic research into cancer radiotherapy and medcine development. PMID:27779109

  17. Functional characterization of CYP2D6 enhancer polymorphisms.

    PubMed

    Wang, Danxin; Papp, Audrey C; Sun, Xiaochun

    2015-03-15

    CYP2D6 metabolizes nearly 25% of clinically used drugs. Genetic polymorphisms cause large inter-individual variability in CYP2D6 enzyme activity and are currently used as biomarker to predict CYP2D6 metabolizer phenotype. Previously, we had identified a region 115 kb downstream of CYP2D6 as enhancer for CYP2D6, containing two completely linked single nucleotide polymorphisms (SNPs), rs133333 and rs5758550, associated with enhanced transcription. However, the enhancer effect on CYP2D6 expression, and the causative variant, remained to be ascertained. To characterize the CYP2D6 enhancer element, we applied chromatin conformation capture combined with the next-generation sequencing (4C assays) and chromatin immunoprecipitation with P300 antibody, in HepG2 and human primary culture hepatocytes. The results confirmed the role of the previously identified enhancer region in CYP2D6 expression, expanding the number of candidate variants to three highly linked SNPs (rs133333, rs5758550 and rs4822082). Among these, only rs5758550 demonstrated regulating enhancer activity in a reporter gene assay. Use of clustered regularly interspaced short palindromic repeats mediated genome editing in HepG2 cells targeting suspected enhancer regions decreased CYP2D6 mRNA expression by 70%, only upon deletion of the rs5758550 region. These results demonstrate robust effects of both the enhancer element and SNP rs5758550 on CYP2D6 expression, supporting consideration of rs5758550 for CYP2D6 genotyping panels to yield more accurate phenotype prediction. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    PubMed

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  19. Functionalized 2D-MoS2-Incorporated Polymer Ternary Solar Cells: Role of Nanosheet-Induced Long-Range Ordering of Polymer Chains on Charge Transport.

    PubMed

    Ahmad, Razi; Srivastava, Ritu; Yadav, Sushma; Chand, Suresh; Sapra, Sameer

    2017-09-19

    In this paper, we demonstrated the enhancement in power conversion efficiency (PCE) of solar cells based on poly(3-hexylthiophene-2,5-diyl) (P3HT)/[6,6]-phenyl C71 butyric acid methyl ester (PC71BM) by incorporation of functionalized 2D-MoS2 nanosheets (NSs) as an additional charge-transporting material. The enhancement in PCE of ternary solar cells arises due to the synergic enhancement in exciton dissociation and the improvement in mobility of both electrons and holes through the active layer of the solar cells. The improved hole mobility is attributed to the formation of the long-range ordered nanofibrillar structure of polymer phases and improved crystallinity in the presence of 2D-MoS2 NSs. The improved electron mobility arises due to the highly conducting 2D network of MoS2 NSs which provides additional electron transport channels within the active layer. The nanosheet-incorporated ternary blend solar cells exhibit 32% enhancement in PCE relative to the binary blend P3HT/PC71BM.

  20. Fractal dimension as a measure of altered actin cytoskeleton in MC3T3-E1 cells under simulated microgravity using 3-D/2-D clinostats.

    PubMed

    Qian, A R; Li, D; Han, J; Gao, X; Di, S M; Zhang, W; Hu, L F; Shang, Peng

    2012-05-01

    Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

  1. Giardia duodenalis: enhanced growth in cell culture.

    PubMed

    Bowie, W R; Isaac-Renton, J L; Prasad, N

    1988-01-01

    Growth of Giardia duodenalis in broth and in animals has been studied in considerable detail. In contrast, the kinetics of growth in cell culture have been little evaluated. In this study, in vitro growth of G. duodenalis was evaluated in cell culture, primarily using mouse McCoy cells in vials. The media used were Giardia broth (TYI-G), Trichomonas vaginalis broth (TYI-T), and standard cell culture media (CMGA) alone and in combination (2 parts by volume CMGA to one part of TYI broth). Addition of cell culture enhanced the sensitivity of the systems in detecting low numbers of G. duodenalis. Growth was identified consistently with inocula less than or equal to 10/ml, and often with a calculated 10-1/ml inoculum with CMGA/TYI-T and CMGA/TYI-G with cells, and with TYI-G with and without cells. The 2 preferred systems for sensitivity and growth were CMGA/TYI-G with cells and TYI-G with cells. The pH fell minimally in the growth systems and, if CMGA was in the media, cell monolayers remained intact and viable throughout the experiment. In preliminary experiments, cell cultures did not allow growth of one strain of G. muris. These cell culture systems may be useful for detection of low numbers of non-laboratory adapted trophozoites, and should be useful in evaluating the interaction of G. dudodenalis with cells in culture.

  2. Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures.

    PubMed

    Valyi-Nagy, Klara; Kormos, Bernadett; Ali, Mohamed; Shukla, Deepak; Valyi-Nagy, Tibor

    2012-01-01

    Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

  3. Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures

    PubMed Central

    Valyi-Nagy, Klara; Kormos, Bernadett; Ali, Mohamed; Shukla, Deepak

    2012-01-01

    Purpose Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. Methods The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. Results We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. Conclusions These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells. PMID:22419851

  4. Three dimensional culture of the murine osteoblastic cell line OCT-1 on collagen coated microcarriers

    NASA Astrophysics Data System (ADS)

    Lau, P.; Hellweg, C. E.; Kirchner, S.; Baumstark-Khan, C.

    2005-08-01

    During long-term space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. Bone loss during space flight is about 1-2% per month. Bone is continually being remodelled under the influence of three types of highly specialized cells. Osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and finally osteocytes preserve the homeostasis of bone formation and resorption. In vitro 3- dimensional cell culture of osteoblastic cell lines on microcarrier beads might be a better model to evaluate changes in bone cell morphology, function and differentiation under influence of spaceflight related factors than the conventional 2-D monolayer culture technique. Furthermore, it allows production of a greater amount of cells compared to the monolayer culture. Aim of this study is to examine the effects of culturing the immortalized murine osteoblastic cell line OCT-1 in a 3- dimensional environment on cell morphology and proliferation rate.

  5. Mean cell size and collagen orientation from 2D Fourier analysis on confocal laser scanning microscopy and two-photon fluorescence microscopy on human skin in vivo

    NASA Astrophysics Data System (ADS)

    Lucassen, Gerald W.; Bakker, Bernard L.; Neerken, Sieglinde; Hendriks, Rob F. M.

    2003-07-01

    We present results from 2D Fourier analysis on 3D stacks of images obtained by confocal laser scanning reflectance microscopy (CLSM) and two-photon fluorescence microscopy (2PM) on human skin in vivo. CLSM images were obtained with a modified commercial system (Vivascope1000, Lucid Inc, excitation wavelength 830 nm) equipped with a piezo-focusing element (350 μm range) for depth positioning of the objective lens. 2PM was performed with a specially designed set-up with excitation wavelength 730 nm. Mean cell size in the epidermal layer and structural orientation in the dermal layer have been determined as a function of depth by 2D Fourier analysis. Fourier analysis on microscopic images enables automatic non-invasive quantitative structural analysis (mean cell size and orientation) of living human skin.

  6. Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation.

    PubMed

    Watson, P Marc D; Kavanagh, Edel; Allenby, Gary; Vassey, Matthew

    2017-02-01

    Neurodegeneration and neuroinflammation are key features in a range of chronic central nervous system (CNS) diseases such as Alzheimer's and Parkinson's disease, as well as acute conditions like stroke and traumatic brain injury, for which there remains significant unmet clinical need. It is now well recognized that current cell culture methodologies are limited in their ability to recapitulate the cellular environment that is present in vivo, and there is a growing body of evidence to show that three-dimensional (3D) culture systems represent a more physiologically accurate model than traditional two-dimensional (2D) cultures. Given the complexity of the environment from which cells originate, and their various cell-cell and cell-matrix interactions, it is important to develop models that can be controlled and reproducible for drug discovery. 3D cell models have now been developed for almost all CNS cell types, including neurons, astrocytes, microglia, and oligodendrocyte cells. This review will highlight a number of current and emerging techniques for the culture of astrocytes and microglia, glial cell types with a critical role in neurodegenerative and neuroinflammatory conditions. We describe recent advances in glial cell culture using electrospun polymers and hydrogel macromolecules, and highlight how these novel culture environments influence astrocyte and microglial phenotypes in vitro, as compared to traditional 2D systems. These models will be explored to illuminate current trends in the techniques used to create 3D environments for application in research and drug discovery focused on astrocytes and microglial cells.

  7. Effect of 1,25(OH)2D3 on transdifferentiation of rat renal tubular epithelial cells induced by high glucose

    PubMed Central

    Hu, Hongtao; Xu, Shen; Hu, Shuang; Gao, Yue; Shui, Hua

    2016-01-01

    Deficiency in vitamin D and its active metabolite is a characteristic of chronic kidney diseases (CKDs). Previous studies have reported that 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D, can attenuate renal interstitial fibrosis. The present study aimed to explore the effect of 1,25(OH)2D3 on the transdifferentiation of NRK-52E rat renal tubular epithelial cells (RTECs) induced by high glucose, as well as the expression of vitamin D receptor (VDR) and production of angiotensin (Ang) II. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses were performed to detect the protein and mRNA expression of α-smooth muscle actin (α-SMA), E-cadherin and VDR. Furthermore, the production of Ang II was analyzed by enzyme-linked immunosorbent assay (ELISA). Treatment with high glucose decreased E-cadherin and VDR, while increasing α-SMA and Ang II, and of note, these changes were attenuated by 1,25(OH)2D3 in a dose-dependent manner. In conclusion, the present study revealed that 1,25(OH)2D3 inhibits high glucose-induced transdifferentiation of rat RTECs in a dose-dependent manner, which may be associated with the downregulation of Ang II and upregulation of VDR. PMID:28101343

  8. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  9. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  10. Fine tuning a well-oiled machine: Influence of NK1.1 and NKG2D on NKT cell development and function

    PubMed Central

    Joshi, Sunil K.; Lang, Mark L.

    2013-01-01

    Natural Killer T cells (NKT) represent a group of CD1d-restricted T-lineage cells that that provide a functional interface between innate and adaptive immune responses in infectious disease, cancer, allergy and autoimmunity. There have been remarkable advances in understanding the molecular events that underpin NKT development in the thymus and in the complex array of functions in the periphery. Most functional studies have focused on activation of T cell antigen receptors expressed by NKT cells and their responses to CD1d presentation of glycolipid and related antigens. Receiving less attention has been several molecules that are hallmarks of Natural Killer (NK) cells, but nonetheless expressed by NKT cells. These include several activating and inhibitory receptors that may fine-tune NKT development and survival, as well as activation via antigen receptors. Herein, we review the possible roles of the NK1.1 and NKG2D receptors in regulating development and function of NKT cells in health and disease. We suggest that pharmacological alteration of NKT activity should consider the potential complexities commensurate with NK1.1 and NKG2D expression. PMID:23800654

  11. Progesterone metabolism in cultured amniotic fluid cells.

    PubMed

    Beling, C G; Cederqvist, L L

    1978-01-01

    Amniotic fluid cells obtained by amnicentesis at 16-20 weeks' gestation were grown in culture until a confluent monolayer of cell had been formed. Radiolabeled pregnenolone, progesterone and 20 alpha-dihydroprogesterone were added to the cell cultures; steroid metabolites which formed after 24 and 48 hours of incubation were identified. Incubation of the cell cultures with pregnenolone-3H resulted in the formation of progesterone, 17alpha-progesterone and 20 alpha-dihydroprogesterone. A significant amount of progesterone was identified after incubating the cell cultures with 20 alpha-dihydroprogesterone. The results indicate that 3 beta-ol-dehydrogenase, 17 alpha-hydroxylase and 20 alpha-hydroxysteroid dehydrogenase enzymes are present in cultured amniotic fluid cells obtained at 16-20 weeks' gestation.

  12. Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

    NASA Technical Reports Server (NTRS)

    Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

    2008-01-01

    Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

  13. Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

    2017-05-01

    Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

  14. Regulation of the expression of MHC class I-related chain A, B (MICA, MICB) via chromatin remodeling and its impact on the susceptibility of leukemic cells to the cytotoxicity of NKG2D-expressing cells.

    PubMed

    Kato, N; Tanaka, J; Sugita, J; Toubai, T; Miura, Y; Ibata, M; Syono, Y; Ota, S; Kondo, T; Asaka, M; Imamura, M

    2007-10-01

    Innate immune cells such as natural killer (NK) cells play a crucial role in antitumor immune responses. NKG2D is a major activating immunoreceptor expressed in not only NK cells but also CD8+ T cells and shows cytotoxicity against tumors by recognizing its ligands major histocompatibility complex class I-related chain A and B (MICA and MICB) on tumor cells. Recently, it has been suggested that NKG2D-mediated cytotoxicity correlates with the expression levels of NKG2D ligands on target cells. In this study, we were able to increase the expression levels of MICA and MICB on leukemic cell lines and patients' leukemic cells by treatment with trichostatin A (TsA), a histone deacetylase (HDAC) inhibitor. Chromatin immunoprecipitation (ChIP) assays revealed that treatment with TsA resulted in increased acetylation of histone H3 and decreased association with HDAC1 at the promoters of MICA and MICB. Intriguingly, upregulation of MICA and MICB by treatment with TsA led to enhancement of the susceptibility of leukemic cells to the cytotoxicity of NKG2D-expressing cells. Our results suggest that regulation of the expression of NKG2D ligands by treatment with chromatin-remodeling drugs may be an attractive strategy for immunotherapy.

  15. Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation.

    PubMed

    Seiler, Christof; Gazdhar, Amiq; Reyes, Mauricio; Benneker, Lorin M; Geiser, Thomas; Siebenrock, Klaus A; Gantenbein-Ritter, Benjamin

    2014-09-01

    Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of

  16. Single-cell growth analysis in a mixed cell culture

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Bato, Mary Grace P.; Daria, Vincent Ricardo

    2008-06-01

    We perform single cell analysis of cell growth in a mixed cell culture. Two species of yeast cells: Saccharomyces cerevisiae and Candida albicans, are optically trapped using focused continuous-wave near infrared laser. Cell growth for both cells is inhibited only when the two species of cells are in contact with each other. This indicates cell-cell interaction mediated cell growth inhibition mechanism. Single cell level analysis of cell growth studied here contributes to the further understanding of yeast growth arrest in a mixed yeast culture.

  17. Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells

    PubMed Central

    Nemere, I.; Farach-Carson, M. C.; Rohe, B.; Sterling, T. M.; Norman, A. W.; Boyan, B. D.; Safford, S. E.

    2004-01-01

    We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)2D3 and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D3-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)2D3 binding, but did not affect binding to the nuclear receptor for 1,25(OH)2D3. Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D3-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)2D3 in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D3-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D3-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class. PMID:15123837

  18. Water-Soluble 2D Transition Metal Dichalcogenides as the Hole-Transport Layer for Highly Efficient and Stable p-i-n Perovskite Solar Cells.

    PubMed

    Huang, Peng; Wang, Zhaowei; Liu, Yanfeng; Zhang, Kaicheng; Yuan, Ligang; Zhou, Yi; Song, Bo; Li, Yongfang

    2017-08-02

    As a hole-transport layer (HTL) material, poly(3,4-ethylenedioxythiophene):polystyrene-sulfonate (PEDOT:PSS) was often criticized for its intrinsic acidity and hygroscopic effect that would in the long run affect the stability of perovskite solar cells (Pero-SCs). As alternatives, herein water-soluble two-dimensional (2D) transition metal dichalcogenides (TMDs), such as MoS2 and WS2 were used as HTLs in p-i-n Pero-SCs. It was found that the content of 1T phase in 2D TMDs HTLs is centrally important to the power conversion efficiencies (PCEs) of Pero-SCs, and the 1T-rich TMDs (as achieved from exfoliation and without postheating) lead to much higher PCEs. More importantly, as PEDOT:PSS was replaced by 2D TMDs, both the PCEs and stability of Pero-SCs were significantly improved. The highest PCEs of 14.35 and 15.00% were obtained for the Pero-SCs with MoS2 and WS2, respectively, whereas the Pero-SCs with PEDOT:PSS showed a highest PCE of only 12.44%. These are up to date the highest PCEs using 2D TMDs as HTLs. After being stored in a glovebox for 56 days, PCEs of the Pero-SCs using MoS2 and WS2 HTLs remained 78 and 72%, respectively, whereas the PCEs of Pero-SCs with PEDOT:PSS almost dropped to 0 over 35 days. This study demonstrates that water-soluble 2D TMDs have great potential for application as new generation of HTLs aiming at high performance and long-term stable Pero-SCs.

  19. 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.

  20. Capabilities using 2-D DIGE in proteomics research : the new gold standard for 2-D gel electrophoresis.

    PubMed

    Rozanas, Christine R; Loyland, Stacey M

    2008-01-01

    The use of two-dimensional gel electrophoresis for differential analysis in proteomics was revolutionized by the introduction of 2-D fluorescence difference gel electrophoresis (2-D DIGE). This fluorescence-based technique allows the use of multiplexed samples and an internal standard that virtually eliminates gel-to-gel variability, resulting in increased confidence that differences found between samples are due to real induced changes, rather than inherent biological variation or experimental variability. 2-D DIGE has quickly become the "gold standard" for gel-based proteomics for studying tissues, as well as cell culture and bodily fluids such as serum or plasma. This chapter will describe the basic 2-D DIGE technique using minimal labeling, image acquisition using high-quality fluorescence scanners, and software capable of analyzing the multiplexed images and normalizing the data using the internal standard.

  1. Data of the recombination loss mechanisms analysis on Al2O3 PERC cell using PC1D and PC2D simulations.

    PubMed

    Huang, Haibing; Lv, Jun; Bao, Yameng; Xuan, Rongwei; Sun, Shenghua; Sneck, Sami; Li, Shuo; Modanese, Chiara; Savin, Hele; Wang, Aihua; Zhao, Jianhua

    2017-04-01

    This data article is related to our recently published article ('20.8% industrial PERC solar cell: ALD Al2O3 rear surface passivation, efficiency loss mechanisms analysis and roadmap to 24%', Huang et al., 2017 [1]) where we have presented a systematic evaluation of the overall cell processing and a cost-efficient industrial roadmap for PERC cells. Aside from the information already presented in Huang et al., 2017 [1], here we provide data related to Sectin 3 in Huang et al., 2017 [1] concerning the analysis of the recombination losses׳ mechanisms by PC1D V5.9 and PC2D simulations (Clugston and Basore, 1997, Basore and Cabanas-Holmen, 2011, Cabanas-Holmen and Basore, 2012 and Cabanas-Holmen and Basore, 2012.) [2], [3], [4], [5] on our current industrial Al2O3 PERC cell. The data include: i) PC2D simulations on J02, ii) the calculation of series resistance and back surface recombination velocity (BSRV) on the rear side metallization of PERC cell for the case of a point contact, and iii) the PC1D simulation on the cumulative photo-generation and recombination along the distance from the front surface. Finally, the roadmap of the solar cell efficiency for an industrial PERC technology up to 24% is presented, with the aim of providing a potential guideline for industrial researchers.

  2. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures....

  3. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures....

  4. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures....

  5. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells.

    PubMed

    Gurel Pekozer, Gorke; Torun Kose, Gamze; Hasirci, Vasif

    2016-11-01

    Co-culture of bone forming cells and endothelial cells to induce pre-vascularization is one of the strategies used to solve the insufficient vascularization problem in bone tissue engineering attempts. In the study, primary cells isolated from 2 different tissues of the same animal, rat bone marrow stem cells (RBMSCs) and rat aortic endothelial cells (RAECs) were co-cultured to study the effects of co-culturing on both osteogenesis and angiogenesis. The formation of tube like structure in 2D culture was observed for the first time in the literature by the co-culture of primary cells from the same animal and also osteogenesis and angiogenesis were investigated at the same time by using this co-culture system. Co-cultured cells mineralized and formed microvasculature beginning from 14days of incubation. After 28days of incubation in the osteogenic medium, expression of osteogenic genes in co-cultures was significantly upregulated compared to RBMSCs cultured alone. These results suggest that the co-culture of endothelial cells with mesenchymal stem cells induces both osteogenesis and angiogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Ligand accumulation in autocrine cell cultures.

    PubMed

    Monine, Michael I; Berezhkovskii, Alexander M; Joslin, Elizabeth J; Wiley, H Steven; Lauffenburger, Douglas A; Shvartsman, Stanislav Y

    2005-04-01

    Cell-culture assays are routinely used to analyze autocrine signaling systems, but quantitative experiments are rarely possible. To enable the quantitative design and analysis of experiments with autocrine cells, we develop a biophysical theory of ligand accumulation in cell-culture assays. Our theory predicts the ligand concentration as a function of time and measurable parameters of autocrine cells and cell-culture experiments. The key step of our analysis is the derivation of the survival probability of a single ligand released from the surface of an autocrine cell. An expression for this probability is derived using the boundary homogenization approach and tested by stochastic simulations. We use this expression in the integral balance equations, from which we find the Laplace transform of the ligand concentration. We demonstrate how the theory works by analyzing the autocrine epidermal growth factor receptor system and discuss the extension of our methods to other experiments with cultured autocrine cells.

  7. A practical guide to microfluidic perfusion culture of adherent mammalian cells.

    PubMed

    Kim, Lily; Toh, Yi-Chin; Voldman, Joel; Yu, Hanry

    2007-06-01

    Culturing cells at microscales allows control over microenvironmental cues, such as cell-cell and cell-matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.

  8. A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses

    PubMed Central

    Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

    2017-01-01

    Abstract Background. Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM. PMID:27576873

  9. A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.

    PubMed

    Gomez-Roman, Natividad; Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

    2017-02-01

    Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug-radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug-radiation combinations for GBM.

  10. Silicon substrate as a novel cell culture device for myoblast cells

    PubMed Central

    2014-01-01

    Background Tissue and organ regeneration via transplantation of cell bodies in-situ has become an interesting strategy in regenerative medicine. Developments of cell carriers to systematically deliver cell bodies in the damage site have fall shorten on effectively meet this purpose due to inappropriate release control. Thus, there is still need of novel substrate to achieve targeted cell delivery with appropriate vehicles. In the present study, silicon based photovoltaic (PV) devices are used as a cell culturing substrate for the expansion of myoblast mouse cell (C2C12 cells) that offers an atmosphere for regular cell growth in vitro. The adherence, viability and proliferation of the cells on the silicon surface were examined by direct cell counting and fluorescence microscopy. Results It was found that on the silicon surface, cells proliferated over 7 days showing normal morphology, and expressed their biological activities. Cell culture on silicon substrate reveals their attachment and proliferation over the surface of the PV device. After first day of culture, cell viability was 88% and cell survival remained above 86% as compared to the seeding day after the seventh day. Furthermore, the DAPI staining revealed that the initially scattered cells were able to eventually build a cellular monolayer on top of the silicon substrate. Conclusions This study explored the biological applications of silicon based PV devices, demonstrating its biocompatibility properties and found useful for culture of cells on porous 2-D surface. The incorporation of silicon substrate has been efficaciously revealed as a potential cell carrier or vehicle in cell growth technology, allowing for their use in cell based gene therapy, tissue engineering, and therapeutic angiogenesis. PMID:24885347

  11. The Volume of Three-Dimensional Cultures of Cancer Cells InVitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors.

    PubMed

    Boghaert, Erwin R; Lu, Xin; Hessler, Paul E; McGonigal, Thomas P; Oleksijew, Anatol; Mitten, Michael J; Foster-Duke, Kelly; Hickson, Jonathan A; Santo, Vitor E; Brito, Catarina; Uziel, Tamar; Vaidya, Kedar S

    2017-09-01

    Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of

  12. Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection

    PubMed Central

    Sainz, Bruno; TenCate, Veronica; Uprichard, Susan L

    2009-01-01

    Background In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells. Results When cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4α, Albumin, TTR and α1AT), were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, β-Catenin and E-Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization. Conclusion These findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system. PMID:19604376

  13. Dynamic culture improves cell reprogramming efficiency.

    PubMed

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

  14. Dynamic Culture Improves Cell Reprogramming Efficiency

    PubMed Central

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-01-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling. PMID:27031931

  15. New culture medium concepts for cell transplantation.

    PubMed

    Lee, S; Kim, B Y; Yeo, J E; Nemeno, J G; Jo, Y H; Yang, W; Nam, B M; Namoto, S; Tanaka, S; Sato, M; Lee, K M; Hwang, H S; Lee, J I

    2013-10-01

    Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications. A modified culture medium--medical fluid-based culture medium (FCM)--was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbecco's modified Eagle's medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells. We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG. Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  16. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  17. Holographic intravital microscopy for 2-