Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

PubMed Central

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A.; Alves, Paula M.

2016-01-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. Significance The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. PMID:27025693

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing.

PubMed

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A; Serra, Margarida; Alves, Paula M

2016-05-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. ©AlphaMed Press.

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

NASA Astrophysics Data System (ADS)

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

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

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

Intrinsic Lens Forming Potential of Mouse Lens Epithelial versus Newt Iris Pigment Epithelial Cells in Three-Dimensional Culture

PubMed Central

Nakamura, Kenta; Tsonis, Panagiotis A.

2014-01-01

Adult newts (Notophthalmus viridescens) are capable of complete lens regeneration that is mediated through dorsal iris pigment epithelial (IPE) cells transdifferentiation. In contrast, higher vertebrates such as mice demonstrate only limited lens regeneration in the presence of an intact lens capsule with remaining lens epithelial cells. To compare the intrinsic lens regeneration potential of newt IPE versus mouse lens epithelial cells (MLE), we have established a novel culture method that uses cell aggregation before culture in growth factor-reduced Matrigel™. Dorsal newt IPE aggregates demonstrated complete lens formation within 1 to 2 weeks of Matrigel culture without basic fibroblast growth factor (bFGF) supplementation, including the establishment of a peripheral cuboidal epithelial cell layer, and the appearance of central lens fibers that were positive for αA-crystallin. In contrast, the lens-forming potential of MLE cell aggregates cultured in Matrigel was incomplete and resulted in the formation of defined-size lentoids with partial optical transparency. While the peripheral cell layers of MLE aggregates were nucleated, cells in the center of aggregates demonstrated a nonapoptotic nuclear loss over a time period of 3 weeks that was representative of lens fiber formation. Matrigel culture supplementation with bFGF resulted in higher transparent bigger-size MLE aggregates that demonstrated increased appearance of βB1-crystallin expression. Our study demonstrates that bFGF is not required for induction of newt IPE aggregate-dependent lens formation in Matrigel, while the addition of bFGF seems to be beneficial for the formation of MLE aggregate-derived lens-like structures. In conclusion, the three-dimensional aggregate culture of IPE and MLE in Matrigel allows to a higher extent than older models the indepth study of the intrinsic lens-forming potential and the corresponding identification of lentogenic factors. PMID:23672748

Modulation of invasive phenotype by interstitial pressure-driven convection in aggregates of human breast cancer cells.

PubMed

Tien, Joe; Truslow, James G; Nelson, Celeste M

2012-01-01

This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion.

Physical confinement signals regulate the organization of stem cells in three dimensions

PubMed Central

Sean, David; Ignacio, Maxime; Godin, Michel; Slater, Gary W.; Pelling, Andrew E.

2016-01-01

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis in vitro, mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell–cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell–substrate versus cell–cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell–cell and cell–substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells. PMID:27798278

Human brain metastatic stroma attracts breast cancer cells via chemokines CXCL16 and CXCL12.

PubMed

Chung, Brile; Esmaeili, Ali A; Gopalakrishna-Pillai, Sailesh; Murad, John P; Andersen, Emily S; Kumar Reddy, Naveen; Srinivasan, Gayathri; Armstrong, Brian; Chu, Caleb; Kim, Young; Tong, Tommy; Waisman, James; Yim, John H; Badie, Behnam; Lee, Peter P

2017-01-01

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor-ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.

Cultured normal mammalian tissue and process

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

1993-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Induction of carcinoembryonic antigen expression in a three-dimensional culture system

NASA Technical Reports Server (NTRS)

Jessup, J. M.; Brown, D.; Fitzgerald, W.; Ford, R. D.; Nachman, A.; Goodwin, T. J.; Spaulding, G.

1994-01-01

MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in vitro in monolayer culture. MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether MIP-101 cells may be induced to express CEA when cultured on microcarrier beads in three-dimensional cultures, either in static cultures as non-adherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP- 101 cells proliferated well under all three conditions and increased CEA and NCA production 3 - 4 fold when grown in three-dimensional cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that three-dimensional growth in vitro simulates tumor function in vivo and that three-dimensional growth by itself may enhance production of molecules that are associated with the metastatic process.

A microfabricated platform to form three-dimensional toroidal multicellular aggregate.

PubMed

Masuda, Taisuke; Takei, Natsuki; Nakano, Takuma; Anada, Takahisa; Suzuki, Osamu; Arai, Fumihito

2012-12-01

Techniques that allow cells to self-assemble into three-dimensional (3D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular in fields such as stem cell research, tissue engineering, and cancer biology. Appropriate simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. We have developed a unique multicellular aggregate formation platform that utilizes a maskless gray-scale photolithography. The cellular aggregate formed using this platform has a toroidal-like geometry and includes a micro lumen that facilitates the supply of oxygen and growth factors and the expulsion of waste products. As a result, this platform was capable of rapidly producing hundreds of multicellular aggregates at a time, and of regulating the diameter of aggregates with complex design. These toroidal multicellular aggregates can grow as long-term culture. In addition, the micro lumen can be used as a continuous channel and for the insertion of a vascular system or a nerve system into the assembled tissue. These platform characteristics highlight its potential to be used in a wide variety of applications, e.g. as a bioactuator, as a micro-machine component or in drug screening and tissue engineering.

Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel

NASA Technical Reports Server (NTRS)

Qiu, Q.; Ducheyne, P.; Gao, H.; Ayyaswamy, P.

1998-01-01

Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chain-like or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

Protein Misfolding Diseases.

PubMed

Hartl, F Ulrich

2017-06-20

The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

PubMed

Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

2017-01-01

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Growing Bladder-Cancer Cells In Three-Dimensional Clusters

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F.; Prewett, Tacey L.; Goodwin, Thomas J.

1995-01-01

Artificial growth process helps fill gaps in cancer research. Cell cultures more accurate as models for in vivo studies and as sources of seed cells for in vivo studies. Effected in horizontal rotating bioreactor described in companion article, "Simplified Bioreactor for Growing Mammalian Cells" (MSC-22060). Provides aggregates of cells needed to fill many of gaps.

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

PubMed

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Dielectric spectroscopy platform to measure MCF10A epithelial cell aggregation as a model for spheroidal cell cluster analysis.

PubMed

Heileman, K L; Tabrizian, M

2017-05-02

3-Dimensional cell cultures are more representative of the native environment than traditional cell cultures on flat substrates. As a result, 3-dimensional cell cultures have emerged as a very valuable model environment to study tumorigenesis, organogenesis and tissue regeneration. Many of these models encompass the formation of cell aggregates, which mimic the architecture of tumor and organ tissue. Dielectric impedance spectroscopy is a non-invasive, label free and real time technique, overcoming the drawbacks of established techniques to monitor cell aggregates. Here we introduce a platform to monitor cell aggregation in a 3-dimensional extracellular matrix using dielectric spectroscopy. The MCF10A breast epithelial cell line serves as a model for cell aggregation. The platform maintains sterile conditions during the multi-day assay while allowing continuous dielectric spectroscopy measurements. The platform geometry optimizes dielectric measurements by concentrating cells within the electrode sensing region. The cells show a characteristic dielectric response to aggregation which corroborates with finite element analysis computer simulations. By fitting the experimental dielectric spectra to the Cole-Cole equation, we demonstrated that the dispersion intensity Δε and the characteristic frequency f c are related to cell aggregate growth. In addition, microscopy can be performed directly on the platform providing information about cell position, density and morphology. This platform could yield many applications for studying the electrophysiological activity of cell aggregates.

Role of Multicellular Aggregates in Biofilm Formation

PubMed Central

Kragh, Kasper N.; Hutchison, Jaime B.; Melaugh, Gavin; Rodesney, Chris; Roberts, Aled E. L.; Irie, Yasuhiko; Jensen, Peter Ø.; Diggle, Stephen P.; Allen, Rosalind J.

2016-01-01

ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. PMID:27006463

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

Physiological and Molecular Genetic Effects of Time-Varying Electromagnetic Fields on Human Neuronal Cells

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.

2003-01-01

The present investigation details the development of model systems for growing two- and three-dimensional human neural progenitor cells within a culture medium facilitated by a time-varying electromagnetic field (TVEMF). The cells and culture medium are contained within a two- or three-dimensional culture vessel, and the electromagnetic field is emitted from an electrode or coil. These studies further provide methods to promote neural tissue regeneration by means of culturing the neural cells in either configuration. Grown in two dimensions, neuronal cells extended longitudinally, forming tissue strands extending axially along and within electrodes comprising electrically conductive channels or guides through which a time-varying electrical current was conducted. In the three-dimensional aspect, exposure to TVEMF resulted in the development of three-dimensional aggregates, which emulated organized neural tissues. In both experimental configurations, the proliferation rate of the TVEMF cells was 2.5 to 4.0 times the rate of the non-waveform cells. Each of the experimental embodiments resulted in similar molecular genetic changes regarding the growth potential of the tissues as measured by gene chip analyses, which measured more than 10,000 human genes simultaneously.

Colony, hanging drop, and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines.

PubMed

Matak, Damian; Brodaczewska, Klaudia K; Lipiec, Monika; Szymanski, Łukasz; Szczylik, Cezary; Czarnecka, Anna M

2017-08-01

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.

Multilayer Dye Aggregation at Dye/TiO2 Interface via π…π Stacking and Hydrogen Bond and Its Impact on Solar Cell Performance: A DFT Analysis.

PubMed

Zhang, Lei; Liu, Xiaogang; Rao, Weifeng; Li, Jingfa

2016-10-21

Multilayer dye aggregation at the dye/TiO 2 interface of dye-sensitized solar cells is probed via first principles calculations, using p-methyl red azo dye as an example. Our calculations suggest that the multilayer dye aggregates at the TiO 2 surface can be stabilized by π…π stacking and hydrogen bond interactions. Compared with previous two-dimensional monolayer dye/TiO 2 model, the multilayer dye aggregation model proposed in this study constructs a three-dimensional multilayer dye/TiO 2 interfacial structure, and provides a better agreement between experimental and computational results in dye coverage and dye adsorption energy. In particular, a dimer forms by π…π stacking interactions between two neighboring azo molecules, while one of them chemisorbs on the TiO 2 surface; a trimer may form by introducing one additional azo molecule on the dimer through a hydrogen bond between two carboxylic acid groups. Different forms of multilayer dye aggregates, either stabilized by π…π stacking or hydrogen bond, exhibit varied optical absorption spectra and electronic properties. Such variations could have a critical impact on the performance of dye sensitized solar cells.

Multilayer Dye Aggregation at Dye/TiO2 Interface via π…π Stacking and Hydrogen Bond and Its Impact on Solar Cell Performance: A DFT Analysis

PubMed Central

Zhang, Lei; Liu, Xiaogang; Rao, Weifeng; Li, Jingfa

2016-01-01

Multilayer dye aggregation at the dye/TiO2 interface of dye-sensitized solar cells is probed via first principles calculations, using p-methyl red azo dye as an example. Our calculations suggest that the multilayer dye aggregates at the TiO2 surface can be stabilized by π…π stacking and hydrogen bond interactions. Compared with previous two-dimensional monolayer dye/TiO2 model, the multilayer dye aggregation model proposed in this study constructs a three-dimensional multilayer dye/TiO2 interfacial structure, and provides a better agreement between experimental and computational results in dye coverage and dye adsorption energy. In particular, a dimer forms by π…π stacking interactions between two neighboring azo molecules, while one of them chemisorbs on the TiO2 surface; a trimer may form by introducing one additional azo molecule on the dimer through a hydrogen bond between two carboxylic acid groups. Different forms of multilayer dye aggregates, either stabilized by π…π stacking or hydrogen bond, exhibit varied optical absorption spectra and electronic properties. Such variations could have a critical impact on the performance of dye sensitized solar cells. PMID:27767196

A system and methodology for high-content visual screening of individual intact living cells in suspension

NASA Astrophysics Data System (ADS)

Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

2007-02-01

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

Data-driven modeling reveals cell behaviors controlling self-organization during Myxococcus xanthus development

PubMed Central

Cotter, Christopher R.; Schüttler, Heinz-Bernd; Igoshin, Oleg A.; Shimkets, Lawrence J.

2017-01-01

Collective cell movement is critical to the emergent properties of many multicellular systems, including microbial self-organization in biofilms, embryogenesis, wound healing, and cancer metastasis. However, even the best-studied systems lack a complete picture of how diverse physical and chemical cues act upon individual cells to ensure coordinated multicellular behavior. Known for its social developmental cycle, the bacterium Myxococcus xanthus uses coordinated movement to generate three-dimensional aggregates called fruiting bodies. Despite extensive progress in identifying genes controlling fruiting body development, cell behaviors and cell–cell communication mechanisms that mediate aggregation are largely unknown. We developed an approach to examine emergent behaviors that couples fluorescent cell tracking with data-driven models. A unique feature of this approach is the ability to identify cell behaviors affecting the observed aggregation dynamics without full knowledge of the underlying biological mechanisms. The fluorescent cell tracking revealed large deviations in the behavior of individual cells. Our modeling method indicated that decreased cell motility inside the aggregates, a biased walk toward aggregate centroids, and alignment among neighboring cells in a radial direction to the nearest aggregate are behaviors that enhance aggregation dynamics. Our modeling method also revealed that aggregation is generally robust to perturbations in these behaviors and identified possible compensatory mechanisms. The resulting approach of directly combining behavior quantification with data-driven simulations can be applied to more complex systems of collective cell movement without prior knowledge of the cellular machinery and behavioral cues. PMID:28533367

Characterizing the Role of Nanoparticle Design on Tumor Transport and Stability in the Extracellular Environment

NASA Astrophysics Data System (ADS)

Albanese, Alexandre

Nanotechnology has emerged as an exciting strategy for the delivery of diagnostic and therapeutic agents into established tumors. Advancements in nanomaterial synthesis have generated an extensive number of nanoparticle designs made from different materials. Unfortunately, it remains impossible to predict a design's effectiveness for in vivo tumor accumulation. Little is known about how a nanoparticle's morphology and surface chemistry affect its interactions with cells and proteins inside the tumor tissue. This thesis focuses on the development of in vitro experimental tools to evaluate how nanoparticle design affects transport in a three-dimensional tumor tissue and stability in the tumor microenvironment. Nanoparticle transport was evaluated using a novel 'tumor-on-a-chip' system where multicellular tumor spheroids were immobilized in a microfluidic channel. This setup created a three-dimensional tumor environment displaying physiological cell density, extracellular matrix organization, and interstitial flow rates. The tumor-on-a-chip demonstrated that accumulation of nanoparticles was limited to diameters below 110 nm and was improved by receptor targeting. Nanoparticle stability in the tumor microenvironment was evaluated using media isolated from different tumor cell lines. Nanoparticle diameter and surface chemistry were important determinants of stability in cancer cell-conditioned media. Small nanoparticles with unstable surface chemistries adsorbed cellular proteins on their surface and were prone to aggregation. Nanoparticle aggregation altered cellular interactions leading to changes in cell uptake. Using a novel technique to generate different aggregate sizes possessing a uniform surface composition, it was determined that aggregation can change receptor affinity, cell internalization mechanisms and sub-cellular sequestration patterns. Data from this thesis characterize the behavior of nanoparticles within modeled tumor environments and provide some preliminary design guidelines for maximizing nanoparticle tumor accumulation. This work highlights the importance of characterizing nano-bio interactions for engineering successful nanomaterial-based delivery systems.

A Two-Dimensional Numerical Investigation of Transport of Malaria-Infected Red Blood Cells in Stenotic Microchannels

PubMed Central

Tao, Yong; Rongin, Uwitije; Xing, Zhongwen

2016-01-01

The malaria-infected red blood cells experience a significant decrease in cell deformability and increase in cell membrane adhesion. Blood hemodynamics in microvessels is significantly affected by the alteration of the mechanical property as well as the aggregation of parasitized red blood cells. In this study, we aim to numerically study the connection between cell-level mechanobiological properties of human red blood cells and related malaria disease state by investigating the transport of multiple red blood cell aggregates passing through microchannels with symmetric stenosis. Effects of stenosis magnitude, aggregation strength, and cell deformability on cell rheology and flow characteristics were studied by a two-dimensional model using the fictitious domain-immersed boundary method. The results indicated that the motion and dissociation of red blood cell aggregates were influenced by these factors and the flow resistance increases with the increase of aggregating strength and cell stiffness. Further, the roughness of the velocity profile was enhanced by cell aggregation, which considerably affected the blood flow characteristics. The study may assist us in understanding cellular-level mechanisms in disease development. PMID:28105411

Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

PubMed Central

Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

2015-01-01

We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures. PMID:26462469

Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

NASA Astrophysics Data System (ADS)

Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

2015-10-01

We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells.

PubMed

Raz, Vered; Vermolen, Bart J; Garini, Yuval; Onderwater, Jos J M; Mommaas-Kienhuis, Mieke A; Koster, Abraham J; Young, Ian T; Tanke, Hans; Dirks, Roeland W

2008-12-15

Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with gamma-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence.

Aggregation of Culture Expanded Human Mesenchymal Stem Cells in Microcarrier-based Bioreactor.

PubMed

Yuan, Xuegang; Tsai, Ang-Chen; Farrance, Iain; Rowley, Jon; Ma, Teng

2018-03-15

Three-dimensional aggregation of human mesenchymal stem cells (hMSCs) has been used to enhance their therapeutic properties but current fabrication protocols depend on laboratory methods and are not scalable. In this study, we developed thermal responsive poly(N-isopropylacrylamide) grafted microcarriers (PNIPAM-MCs), which supported expansion and thermal detachment of hMSCs at reduced temperature (23.0 °C). hMSCs were cultured on the PNIPAM-MCs in both spinner flask (SF) and PBS Vertical-Wheel (PBS-VW) bioreactors for expansion. At room temperature, hMSCs were detached as small cell sheets, which subsequently self-assembled into 3D hMSC aggregates in PBS-VW bioreactor and remain as single cells in SF bioreactor owing to different hydrodynamic conditions. hMSC aggregates generated from the bioreactor maintained comparable immunomodulation and cytokine secretion properties compared to the ones made from the AggreWell ® . The results of the current study demonstrate the feasibility of scale-up production of hMSC aggregates in the suspension bioreactor using thermal responsive microcarriers for integrated cell expansion and 3D aggregation in a close bioreactor system and highlight the critical role of hydrodynamics in self-assembly of detached hMSC in suspension.

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

PubMed

Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-12-01

In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

Study of the cell activity in three-dimensional cell culture by using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Arunngam, Pakajiraporn; Mahardika, Anggara; Hiroko, Matsuyoshi; Andriana, Bibin Bintang; Tabata, Yasuhiko; Sato, Hidetoshi

2018-02-01

The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm-1 which assigned to PO43- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.

Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems

NASA Astrophysics Data System (ADS)

Flenner, Elijah; Janosi, Lorant; Barz, Bogdan; Neagu, Adrian; Forgacs, Gabor; Kosztin, Ioan

2012-03-01

Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

Hydrofocusing Bioreactor Produces Anti-Cancer Alkaloids

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Valluri, Jagan V.

2011-01-01

A methodology for growing three-dimensional plant tissue models in a hydrodynamic focusing bioreactor (HFB) has been developed. The methodology is expected to be widely applicable, both on Earth and in outer space, as a means of growing plant cells and aggregates thereof under controlled conditions for diverse purposes, including research on effects of gravitation and other environmental factors upon plant growth and utilization of plant tissue cultures to produce drugs in quantities greater and at costs lower than those of conventional methodologies. The HFB was described in Hydro focus - ing Bioreactor for Three-Dimensional Cell Culture (MSC-22358), NASA Tech Briefs, Vol. 27, No. 3 (March 2003), page 66. To recapitulate: The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear liquid culture environment simultaneously with the herding of suspended cells and tissue assemblies and removal of unwanted air bubbles. The HFB includes a rotating cell-culture vessel with a centrally located sampling port and an internal rotating viscous spinner attached to a rotating base. The vessel and viscous spinner can be made to rotate at the same speed and direction or different speeds and directions to tailor the flow field and the associated hydrodynamic forces in the vessel in order to obtain low-shear suspension of cells and control of the locations of cells and air bubbles. For research and pharmaceutical-production applications, the HFB offers two major benefits: low shear stress, which promotes the assembly of cells into tissue-like three-dimensional constructs; and randomization of gravitational vectors relative to cells, which affects production of medicinal compounds. Presumably, apposition of plant cells in the absence of shear forces promotes cell-cell contacts, cell aggregation, and cell differentiation. Only gentle mixing is necessary for distributing nutrients and oxygen. It has been postulated that inasmuch as cells in the simulated microgravitation of an HFB do not need to maintain the same surface forces as in normal Earth gravitation, they can divert more energy sources to growth and differentiation and, perhaps, to biosynthesis of greater quantities of desired medicinal compounds. Because one can adjust the HFB to vary effective gravitation, one can also test the effects of intermediate levels of gravitation on biosynthesis of various products. The potential utility of this methodology for producing drugs was demonstrated in experiments in which sandalwood and Madagascar periwinkle cells were grown in an HFB. The conditions in the HFB were chosen to induce the cells to form into aggregate cultures that produced anti-cancer indole alkaloids in amounts greater than do comparable numbers of cells of the same species cultured according to previously known methodologies. The observations made in these experiments were interpreted as suggesting that the aggregation of the cells might be responsible for the enhancement of production of alkaloids.

In vitro differentiation of mouse embryonic stem (mES) cells using the hanging drop method.

PubMed

Wang, Xiang; Yang, Phillip

2008-07-23

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease. When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells.

Production of Normal Mammalian Organ Culture Using a Medium Containing Mem-Alpha, Leibovitz L 15, Glucose Galactose Fructose

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor)

1999-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under micro- gravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel. The medium used for culturing the cells, especially a mixture of epithelial and mesenchymal cells contains a mixture of Mem-alpha and Leibovits L15 supplemented with glucose, galactose and fructose.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

PubMed

Widbiller, M; Lindner, S R; Buchalla, W; Eidt, A; Hiller, K-A; Schmalz, G; Galler, K M

2016-03-01

Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

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

PubMed Central

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

2016-01-01

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

In vitro spatially organizing the differentiation in individual multicellular stem cell aggregates.

PubMed

Qi, Hao; Huang, Guoyou; Han, Yu Long; Lin, Wang; Li, Xiujun; Wang, Shuqi; Lu, Tian Jian; Xu, Feng

2016-01-01

With significant potential as a robust source to produce specific somatic cells for regenerative medicine, stem cells have attracted increasing attention from both academia and government. In vivo, stem cell differentiation is a process under complicated regulations to precisely build tissue with unique spatial structures. Since multicellular spheroidal aggregates of stem cells, commonly called as embryoid bodies (EBs), are considered to be capable of recapitulating the events in early stage of embryonic development, a variety of methods have been developed to form EBs in vitro for studying differentiation of embryonic stem cells. The regulation of stem cell differentiation is crucial in directing stem cells to build tissue with the correct spatial architecture for specific functions. However, stem cells within the three-dimensional multicellular aggregates undergo differentiation in a less unpredictable and spatially controlled manner in vitro than in vivo. Recently, various microengineering technologies have been developed to manipulate stem cells in vitro in a spatially controlled manner. Herein, we take the spotlight on these technologies and researches that bring us the new potential for manipulation of stem cells for specific purposes.

Extending hepatocyte functionality for drug-testing applications using high-viscosity alginate-encapsulated three-dimensional cultures in bioreactors.

PubMed

Miranda, Joana P; Rodrigues, Armanda; Tostões, Rui M; Leite, Sofia; Zimmerman, Heiko; Carrondo, Manuel J T; Alves, Paula M

2010-12-01

The maintenance of differentiated hepatocyte phenotype in vitro depends on several factors-in particular, on extracellular matrix interactions, for example, with three-dimensional (3D) matrices. Alginate hydrogel provides the cells with a good extracellular matrix due to the formation of a massive capsule with semi-permeable properties that allows for diffusion of the medium components into the cells as well as efficient waste product elimination. Simultaneously, alginate protects the cells from shear stress caused by the hydrodynamics when cultured in stirred systems such as bioreactors. We have previously developed a hepatocyte aggregate 3D culture system in a bioreactor where improved hepatocyte functionality could be maintained over longer periods (21 days). In this work, ultra-high-viscosity alginate was used for hepatocyte aggregates entrapment. Hepatocyte biotransformation (phase I and II enzymes), CYP450 inducibility, and secretory capacity (albumin and urea production) were monitored. The analyses were performed in both spinner vessels and bioreactors to test the effect of the pO(2) control, unavailable in the spinners. Performance of alginate-encapsulated hepatocyte aggregates in culture was compared with nonencapsulated aggregate cultures in both bioreactor (controlled environment) and spinner vessels. For both culture systems, hepatocytes' metabolic and biotransformation capacities were maintained for up to 1 month, and encapsulated cells in bioreactors showed the best performance. In particular, albumin production rate increased 2- and 1.5-fold in encapsulated aggregates compared with nonencapsulated aggregates in bioreactor and spinner vessels, respectively. Urea production rate increased twofold in encapsulated cultures compared with nonencapsulated cells, in both bioreactor and spinner vessels. Similarly, in both the bioreactor and the spinner system, cell encapsulation resulted in a 1.5- and 2.8-fold improvement of hepatocyte 7-ethoxycoumarin and uridine diphosphate glucuronosyltransferases (UGT) activities, respectively. For all parameters, but for UGT activity, the bioreactor system resulted better than the spinner vessels; for UGT activity no difference was observed between the two. Furthermore, both encapsulated and nonencapsulated 3D culture systems were inducible by 3-methylcholanthrene and dexamethasone. The encapsulated systems consistently showed improved performance over the nonencapsulated cells, indicating that the protection conferred by the alginate matrix plays a relevant role in maintaining the hepatocyte functionalities in vitro.

Interwoven four-compartment capillary membrane technology for three-dimensional perfusion with decentralized mass exchange to scale up embryonic stem cell culture.

PubMed

Gerlach, Jörg C; Lübberstedt, Marc; Edsbagge, Josefina; Ring, Alexander; Hout, Mariah; Baun, Matt; Rossberg, Ingrid; Knöspel, Fanny; Peters, Grant; Eckert, Klaus; Wulf-Goldenberg, Annika; Björquist, Petter; Stachelscheid, Harald; Urbaniak, Thomas; Schatten, Gerald; Miki, Toshio; Schmelzer, Eva; Zeilinger, Katrin

2010-01-01

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent 'arteriovenous' flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 x 10(9) cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining 'stemness' markers during cell expansion. Copyright 2010 S. Karger AG, Basel.

Modeling and numerical simulations of growth and morphologies of three dimensional aggregated silver films

NASA Astrophysics Data System (ADS)

Davis, L. J.; Boggess, M.; Kodpuak, E.; Deutsch, M.

2012-11-01

We report on a model for the deposition of three dimensional, aggregated nanocrystalline silver films, and an efficient numerical simulation method developed for visualizing such structures. We compare our results to a model system comprising chemically deposited silver films with morphologies ranging from dilute, uniform distributions of nanoparticles to highly porous aggregated networks. Disordered silver films grown in solution on silica substrates are characterized using digital image analysis of high resolution scanning electron micrographs. While the latter technique provides little volume information, plane-projected (two dimensional) island structure and surface coverage may be reliably determined. Three parameters governing film growth are evaluated using these data and used as inputs for the deposition model, greatly reducing computing requirements while still providing direct access to the complete (bulk) structure of the films throughout the growth process. We also show how valuable three dimensional characteristics of the deposited materials can be extracted using the simulated structures.

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

PubMed

Chen, Yu-Chih; Yoon, Euisik

2017-01-01

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

PubMed

Ikeda, Kazuhiro; Nagata, Shogo; Okitsu, Teru; Takeuchi, Shoji

2017-06-06

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

PubMed Central

Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

2011-01-01

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

A model for the kinetics of homotypic cellular aggregation under static conditions

NASA Technical Reports Server (NTRS)

Neelamegham, S.; Munn, L. L.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

1997-01-01

We present the formulation and testing of a mathematical model for the kinetics of homotypic cellular aggregation. The model considers cellular aggregation under no-flow conditions as a two-step process. Individual cells and cell aggregates 1) move on the tissue culture surface and 2) collide with other cells (or aggregates). These collisions lead to the formation of intercellular bonds. The aggregation kinetics are described by a system of coupled, nonlinear ordinary differential equations, and the collision frequency kernel is derived by extending Smoluchowski's colloidal flocculation theory to cell migration and aggregation on a two-dimensional surface. Our results indicate that aggregation rates strongly depend upon the motility of cells and cell aggregates, the frequency of cell-cell collisions, and the strength of intercellular bonds. Model predictions agree well with data from homotypic lymphocyte aggregation experiments using Jurkat cells activated by 33B6, an antibody to the beta 1 integrin. Since cell migration speeds and all the other model parameters can be independently measured, the aggregation model provides a quantitative methodology by which we can accurately evaluate the adhesivity and aggregation behavior of cells.

The nitric oxide donor S-nitrosoglutathione reduces apoptotic primary liver cell loss in a three-dimensional perfusion bioreactor culture model developed for liver support.

PubMed

Prince, Jose M; Vodovotz, Yoram; Baun, Matthew J; Monga, Satdarshan Pal; Billiar, Timothy R; Gerlach, Jörg C

2010-03-01

Artificial extracorporeal support for hepatic failure has met with limited clinical success. In hepatocytes, nitric oxide (NO) functions as an antiapoptotic modulator in response to a variety of stresses. We hypothesized that NO administration would yield improved viability and hepatocellular restructuring in a four-compartment, hollow fiber-based bioreactor with integral oxygenation for dynamic three-dimensional perfusion of hepatic cells in bioartificial liver support systems. Isolated adult rat liver cells were placed in culture medium alone (control) or medium supplemented with various concentrations of an NO donor (S-nitrosoglutathione [GSNO]) in the bioreactors. Media samples were obtained from the cell perfusion circuit to monitor cellular response. After 24 and 72 h, histology biopsies were taken to investigate spontaneous restructuring of the cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to quantify apoptotic nuclei. Control bioreactors exhibited 47.9 +/- 2.9% (mean +/- standard error of the mean) apoptotic nuclei. In contrast, NO-treated bioreactors exhibited a biphasic response. Fewer apoptotic nuclei were seen in the 200 and 500 microM GSNO groups (14.4 +/- 0.4%). No effect was observed in the 10 microM GSNO group (47.3%), and increased TUNEL staining was observed in the 1000 microM GSNO group (82.6%). Media lactate dehydrogenase levels were lower in bioreactor groups treated with 200 or 500 microM GSNO (310 +/- 38 IU/L) compared with the control group (919 +/- 188 IU/L; p < 0.05). Protein synthesis was not affected, as measured by albumin levels in the media (115 +/- 19 microg/day/cell inoculum in GSNO-treated bioreactors at 24 h vs. 110 +/- 13 in controls; p = 0.851). Histologically, all of the bioreactor groups exhibited liver cell aggregates with some attached to the bioreactor capillaries. Increased numbers of cells in the aggregates and superior spontaneous restructuring of the cells were seen at 24 and 72 h in the bioreactor groups treated with either 200 or 500 microM GSNO compared with the control groups. Addition of an NO donor reduces adult rat liver cell apoptosis during the initial 24 h after cell inoculation within a three-dimensional perfusion bioreactor system for liver support and promotes liver cell aggregation and spontaneous restructuring of the cells at 24 and 72 h. GSNO-treated bioreactors remain metabolically active and show significantly lower levels of cellular injury as compared with controls. Further studies will be required to evaluate the impact of NO treatment of liver support bioreactors for clinical studies.

Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

PubMed

Yan, Yuanwei; Sart, Sébastien; Calixto Bejarano, Fabian; Muroski, Megan E; Strouse, Geoffrey F; Grant, Samuel C; Li, Yan

2015-01-01

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers.

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

PubMed Central

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

2014-01-01

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

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets

PubMed Central

Nowicka, Malgorzata; Krieg, Carsten; Weber, Lukas M.; Hartmann, Felix J.; Guglietta, Silvia; Becher, Burkhard; Levesque, Mitchell P.; Robinson, Mark D.

2017-01-01

High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals). PMID:28663787

EDTA enhances high-throughput two-dimensional bioprinting by inhibiting salt scaling and cell aggregation at the nozzle surface.

PubMed

Parzel, Cheryl A; Pepper, Matthew E; Burg, Timothy; Groff, Richard E; Burg, Karen J L

2009-06-01

Tissue-engineering strategies may be employed in the development of in vitro breast tissue models for use in testing regimens of drug therapies and vaccines. The physical and chemical interactions that occur among cells and extracellular matrix components can also be elucidated with these models to gain an understanding of the progression of transformed epithelial cells into tumours and the ultimate metastases of tumour cells. The modified inkjet printer may be a useful tool for creating three-dimensional (3D) in vitro models, because it offers an inexpensive and high-throughput solution to microfabrication, and because the printer can be easily manipulated to produce varying tissue attributes. We hypothesized, however, that when ink is replaced with a biologically based fluid (i.e. a 'bio-ink'), specifically a serum-free cell culture medium, printer nozzle failure can result from salt scale build-up as fluid evaporates on the printhead surface. In this study, ethylene diamine tetra-acetic acid (EDTA) was used as a culture medium additive to prevent salt scaling and cell aggregation during the bioprinting process. The results showed that EDTA, at a concentration typically found in commercially available trypsin solutions (0.53 mM), prevented nozzle failure when a serum-free culture medium was printed from a nozzle at 1000 drops/s. Furthermore, increasing concentrations of EDTA appeared to mildly decrease aggregation of 4T07 cells. Cell viability studies were performed to demonstrate that addition of EDTA did not result in significant cell death. In conclusion, it is recommended that EDTA be incorporated into bio-ink solutions containing salts that could lead to nozzle failure.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy.

PubMed

Gualda, Emilio J; Simão, Daniel; Pinto, Catarina; Alves, Paula M; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

PubMed Central

Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

NASA Technical Reports Server (NTRS)

Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

2003-01-01

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Attempt to develop taste bud models in three-dimensional culture.

PubMed

Nishiyama, Miyako; Yuki, Saori; Fukano, Chiharu; Sako, Hideyuki; Miyamoto, Takenori; Tomooka, Yasuhiro

2011-09-01

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

Dielectric aggregation kinetics of cells in a uniform AC electric field.

PubMed

Tada, Shigeru; Natsuya, Tomoyuki; Tsukamoto, Akira

2014-01-01

Cell manipulation and separation technologies have potential biological and medical applications, including advanced clinical protocols such as tissue engineering. An aggregation model was developed for a human carcinoma (HeLa) cell suspension exposed to a uniform AC electric field, in order to explore the field-induced structure formation and kinetics of cell aggregates. The momentum equations of cells under the action of the dipole-dipole interaction were solved theoretically and the total time required to form linear string-like cluster was derived. The results were compared with those of a numerical simulation. Experiments using HeLa cells were also performed for comparison. The total time required to form linear string-like clusters was derived from a simple theoretical model of the cell cluster kinetics. The growth rates of the average string length of cell aggregates showed good agreement with those of the numerical simulation. In the experiment, cells were found to form massive clusters on the bottom of a chamber. The results imply that the string-like cluster grows rapidly by longitudinal attraction when the electric field is first applied and that this process slows at later times and is replaced by lateral coagulation of short strings. The findings presented here are expected to enable design of methods for the organization of three-dimensional (3D) cellular structures without the use of micro-fabricated substrates, such as 3D biopolymer scaffolds, to manipulate cells into spatial arrangement.

Microgravity

NASA Image and Video Library

2001-06-01

Cells cultured on Earth (left) typically settle quickly on the bottom of culture vessels due to gravity. In microgravity (right), cells remain suspended and aggregate to form three-dimensional tissue. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Bioreactor principles

NASA Technical Reports Server (NTRS)

2001-01-01

Cells cultured on Earth (left) typically settle quickly on the bottom of culture vessels due to gravity. In microgravity (right), cells remain suspended and aggregate to form three-dimensional tissue. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

PubMed

Correia, Cláudia; Koshkin, Alexey; Duarte, Patrícia; Hu, Dongjian; Carido, Madalena; Sebastião, Maria J; Gomes-Alves, Patrícia; Elliott, David A; Domian, Ibrahim J; Teixeira, Ana P; Alves, Paula M; Serra, Margarida

2018-03-01

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs. © 2017 Wiley Periodicals, Inc.

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells.

PubMed

Ueda, Kaori; Onishi, Akishi; Ito, Shin-Ichiro; Nakamura, Makoto; Takahashi, Masayo

2018-01-22

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins. Retinal organoids differentiated from mouse Nrl-eGFP iPS cells were cultured in various mediums during photoreceptor development. To promote rod photoreceptor development, organoids were maintained in media containing 9-cis retinoic acids (9cRA). To obtain retinal organoids with M-opsin expression, we cultured in medium with 1% fetal bovine serum (FBS) supplemented with T3, BMP4, and DAPT. Section immunohistochemistry was performed to visualize the expression of photoreceptor markers. In three-dimensional (3D) retinas exposed to 9cRA, rhodopsin was expressed earlier and S-cone opsins were suppressed. We could maintain 3D retinas up to DD 35 in culture media with 1% FBS. The 3D retinas expressed rhodopsin, S- and M-opsins, but most cone photoreceptors expressed either S- or M-opsins. By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas. Copyright © 2017 Elsevier Inc. All rights reserved.

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.

PubMed

Zhou, Jin; Zhang, Ye; Lin, Qiuxia; Liu, Zhiqiang; Wang, Haibin; Duan, Cuimi; Wang, Yanmeng; Hao, Tong; Wu, Kuiwu; Wang, Changyong

2010-07-01

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Engineering ellipsoidal cap-like hydrogel particles as building blocks or sacrificial templates for three-dimensional cell culture.

PubMed

Zhang, Weiwei; Huang, Guoyou; Ng, Kelvin; Ji, Yuan; Gao, Bin; Huang, Liqing; Zhou, Jinxiong; Lu, Tian Jian; Xu, Feng

2018-03-26

Hydrogel particles that can be engineered to compartmentally culture cells in a three-dimensional (3D) and high-throughput manner have attracted increasing interest in the biomedical area. However, the ability to generate hydrogel particles with specially designed structures and their potential biomedical applications need to be further explored. This work introduces a method for fabricating hydrogel particles in an ellipsoidal cap-like shape (i.e., ellipsoidal cap-like hydrogel particles) by employing an open-pore anodic aluminum oxide membrane. Hydrogel particles of different sizes are fabricated. The ability to produce ellipsoidal cap-like magnetic hydrogel particles with controlled distribution of magnetic nanoparticles is demonstrated. Encapsulated cells show high viability, indicating the potential for using these hydrogel particles as structure- and remote-controllable building blocks for tissue engineering application. Moreover, the hydrogel particles are also used as sacrificial templates for fabricating ellipsoidal cap-like concave wells, which are further applied for producing size controllable cell aggregates. The results are beneficial for the development of hydrogel particles and their applications in 3D cell culture.

A three-dimensional human neural cell culture model of Alzheimer's disease.

PubMed

Choi, Se Hoon; Kim, Young Hye; Hebisch, Matthias; Sliwinski, Christopher; Lee, Seungkyu; D'Avanzo, Carla; Chen, Hechao; Hooli, Basavaraj; Asselin, Caroline; Muffat, Julien; Klee, Justin B; Zhang, Can; Wainger, Brian J; Peitz, Michael; Kovacs, Dora M; Woolf, Clifford J; Wagner, Steven L; Tanzi, Rudolph E; Kim, Doo Yeon

2014-11-13

Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

Induction of three-dimensional assembly of human liver cells by simulated microgravity

NASA Technical Reports Server (NTRS)

Khaoustov, V. I.; Darlington, G. J.; Soriano, H. E.; Krishnan, B.; Risin, D.; Pellis, N. R.; Yoffe, B.

1999-01-01

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Three-Dimensional Bioreactor Technologies for the Cocultivation of Human Mesenchymal Stem/Stromal Cells and Beta Cells

PubMed Central

Petry, Florian; Weidner, Tobias; Salzig, Denise

2018-01-01

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess engineering of the cocultivation processes is necessary to achieve successful beta cell therapy. PMID:29731775

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PubMed

Eguchi, Takanori; Sogawa, Chiharu; Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-Ichi; Okamoto, Kuniaki; Calderwood, Stuart K

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

PubMed Central

Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-ichi; Okamoto, Kuniaki; Calderwood, Stuart K.

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. PMID:29415026

Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

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

Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu

2010-10-15

UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellularmore » spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.« less

Rheology of three-dimensional packings of aggregates: microstructure and effects of nonconvexity.

PubMed

Azéma, Emilien; Radjaï, Farhang; Saint-Cyr, Baptiste; Delenne, Jean-Yves; Sornay, Philippe

2013-05-01

We use three-dimensional contact dynamics simulations to analyze the rheological properties of granular materials composed of rigid aggregates. The aggregates are made from four overlapping spheres and described by a nonconvexity parameter depending on the relative positions of the spheres. The macroscopic and microstructural properties of several sheared packings are analyzed as a function of the degree of nonconvexity of the aggregates. We find that the internal angle of friction increases with the nonconvexity. In contrast, the packing fraction first increases to a maximum value but declines as the nonconvexity increases further. At a high level of nonconvexity, the packings are looser but show a higher shear strength. At the microscopic scale, the fabric and force anisotropy, as well as the friction mobilization, are enhanced by multiple contacts between aggregates and interlocking, thus revealings the mechanical and geometrical origins of shear strength.

Rigidity of transmembrane proteins determines their cluster shape

NASA Astrophysics Data System (ADS)

Jafarinia, Hamidreza; Khoshnood, Atefeh; Jalali, Mir Abbas

2016-01-01

Protein aggregation in cell membrane is vital for the majority of biological functions. Recent experimental results suggest that transmembrane domains of proteins such as α -helices and β -sheets have different structural rigidities. We use molecular dynamics simulation of a coarse-grained model of protein-embedded lipid membranes to investigate the mechanisms of protein clustering. For a variety of protein concentrations, our simulations under thermal equilibrium conditions reveal that the structural rigidity of transmembrane domains dramatically affects interactions and changes the shape of the cluster. We have observed stable large aggregates even in the absence of hydrophobic mismatch, which has been previously proposed as the mechanism of protein aggregation. According to our results, semiflexible proteins aggregate to form two-dimensional clusters, while rigid proteins, by contrast, form one-dimensional string-like structures. By assuming two probable scenarios for the formation of a two-dimensional triangular structure, we calculate the lipid density around protein clusters and find that the difference in lipid distribution around rigid and semiflexible proteins determines the one- or two-dimensional nature of aggregates. It is found that lipids move faster around semiflexible proteins than rigid ones. The aggregation mechanism suggested in this paper can be tested by current state-of-the-art experimental facilities.

Three-dimensional neural differentiation of embryonic stem cells with ACM induction in microfibrous matrices in bioreactors.

PubMed

Liu, Ning; Ouyang, Anli; Li, Yan; Yang, Shang-Tian

2013-01-01

The clinical use of pluripotent stem cell (PSC)-derived neural cells requires an efficient differentiation process for mass production in a bioreactor. Toward this goal, neural differentiation of murine embryonic stem cells (ESCs) in three-dimensional (3D) polyethylene terephthalate microfibrous matrices was investigated in this study. To streamline the process and provide a platform for process integration, the neural differentiation of ESCs was induced with astrocyte-conditioned medium without the formation of embryoid bodies, starting from undifferentiated ESC aggregates expanded in a suspension bioreactor. The 3D neural differentiation was able to generate a complex neural network in the matrices. When compared to 2D differentiation, 3D differentiation in microfibrous matrices resulted in a higher percentage of nestin-positive cells (68% vs. 54%) and upregulated gene expressions of nestin, Nurr1, and tyrosine hydroxylase. High purity of neural differentiation in 3D microfibrous matrix was also demonstrated in a spinner bioreactor with 74% nestin + cells. This study demonstrated the feasibility of a scalable process based on 3D differentiation in microfibrous matrices for the production of ESC-derived neural cells. © 2013 American Institute of Chemical Engineers.

Division of labour and the evolution of multicellularity

PubMed Central

Ispolatov, Iaroslav; Ackermann, Martin; Doebeli, Michael

2012-01-01

Understanding the emergence and evolution of multicellularity and cellular differentiation is a core problem in biology. We develop a quantitative model that shows that a multicellular form emerges from genetically identical unicellular ancestors when the compartmentalization of poorly compatible physiological processes into component cells of an aggregate produces a fitness advantage. This division of labour between the cells in the aggregate occurs spontaneously at the regulatory level owing to mechanisms present in unicellular ancestors and does not require any genetic predisposition for a particular role in the aggregate or any orchestrated cooperative behaviour of aggregate cells. Mathematically, aggregation implies an increase in the dimensionality of phenotype space that generates a fitness landscape with new fitness maxima, in which the unicellular states of optimized metabolism become fitness saddle points. Evolution of multicellularity is modelled as evolution of a hereditary parameter: the propensity of cells to stick together, which determines the fraction of time a cell spends in the aggregate form. Stickiness can increase evolutionarily owing to the fitness advantage generated by the division of labour between cells in an aggregate. PMID:22158952

A 3D human neural cell culture system for modeling Alzheimer’s disease

PubMed Central

Kim, Young Hye; Choi, Se Hoon; D’Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J.; Klee, Justin B.; Brüstle, Oliver; Tanzi, Rudolph E.; Kim, Doo Yeon

2015-01-01

Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer’s disease (AD) because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel three-dimensional (3D) culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of β-amyloid and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix, and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 days. The aggregation of β-amyloid is observed after 6-weeks of differentiation followed by robust tau pathology after 10–14 weeks. PMID:26068894

Three-dimensional prostate tumor model based on a hyaluronic acid-alginate hydrogel for evaluation of anti-cancer drug efficacy.

PubMed

Tang, Yadong; Huang, Boxin; Dong, Yuqin; Wang, Wenlong; Zheng, Xi; Zhou, Wei; Zhang, Kun; Du, Zhiyun

2017-10-01

In vitro cell-based assays are widely applied to evaluate anti-cancer drug efficacy. However, the conventional approaches are mostly based on two-dimensional (2D) culture systems, making it difficult to recapitulate the in vivo tumor scenario because of spatial limitations. Here, we develop an in vitro three-dimensional (3D) prostate tumor model based on a hyaluronic acid (HA)-alginate hybrid hydrogel to bridge the gap between in vitro and in vivo anticancer drug evaluations. In situ encapsulation of PCa cells was achieved by mixing HA and alginate aqueous solutions in the presence of cells and then crosslinking with calcium ions. Unlike in 2D culture, cells were found to aggregate into spheroids in a 3D matrix. The expression of epithelial to mesenchyme transition (EMT) biomarkers was found to be largely enhanced, indicating an increased invasion and metastasis potential in the hydrogel matrix. A significant up-regulation of proangiogenic growth factors (IL-8, VEGF) and matrix metalloproteinases (MMPs) was observed in 3D-cultured PCa cells. The results of anti-cancer drug evaluation suggested a higher drug tolerance within the 3D tumor model compared to conventional 2D-cultured cells. Finally, we found that the drug effect within the in vitro 3D cancer model based on HA-alginate matrix exhibited better predictability for in vivo drug efficacy.

The use of total human bone marrow fraction in a direct three-dimensional expansion approach for bone tissue engineering applications: focus on angiogenesis and osteogenesis.

PubMed

Guerrero, Julien; Oliveira, Hugo; Catros, Sylvain; Siadous, Robin; Derkaoui, Sidi-Mohammed; Bareille, Reine; Letourneur, Didier; Amédée, Joëlle

2015-03-01

Current approaches in bone tissue engineering have shown limited success, mostly owing to insufficient vascularization of the construct. A common approach consists of co-culture of endothelial cells and osteoblastic cells. This strategy uses cells from different sources and differentiation states, thus increasing the complexity upstream of a clinical application. The source of reparative cells is paramount for the success of bone tissue engineering applications. In this context, stem cells obtained from human bone marrow hold much promise. Here, we analyzed the potential of human whole bone marrow cells directly expanded in a three-dimensional (3D) polymer matrix and focused on the further characterization of this heterogeneous population and on their ability to promote angiogenesis and osteogenesis, both in vitro and in vivo, in a subcutaneous model. Cellular aggregates were formed within 24 h and over the 12-day culture period expressed endothelial and bone-specific markers and a specific junctional protein. Ectopic implantation of the tissue-engineered constructs revealed osteoid tissue and vessel formation both at the periphery and within the implant. This work sheds light on the potential clinical use of human whole bone marrow for bone regeneration strategies, focusing on a simplified approach to develop a direct 3D culture without two-dimensional isolation or expansion.

Three-Dimensional Bioprinting for Regenerative Dentistry and Craniofacial Tissue Engineering.

PubMed

Obregon, F; Vaquette, C; Ivanovski, S; Hutmacher, D W; Bertassoni, L E

2015-09-01

Craniofacial tissues are organized with complex 3-dimensional (3D) architectures. Mimicking such 3D complexity and the multicellular interactions naturally occurring in craniofacial structures represents one of the greatest challenges in regenerative dentistry. Three-dimensional bioprinting of tissues and biological structures has been proposed as a promising alternative to address some of these key challenges. It enables precise manufacture of various biomaterials with complex 3D architectures, while being compatible with multiple cell sources and being customizable to patient-specific needs. This review describes different 3D bioprinting methods and summarizes how different classes of biomaterials (polymer hydrogels, ceramics, composites, and cell aggregates) may be used for 3D biomanufacturing of scaffolds, as well as craniofacial tissue analogs. While the fabrication of scaffolds upon which cells attach, migrate, and proliferate is already in use, printing of all the components that form a tissue (living cells and matrix materials together) to produce tissue constructs is still in its early stages. In summary, this review seeks to highlight some of the key advantages of 3D bioprinting technology for the regeneration of craniofacial structures. Additionally, it stimulates progress on the development of strategies that will promote the translation of craniofacial tissue engineering from the laboratory bench to the chair side. © International & American Associations for Dental Research 2015.

Electrospun polystyrene scaffolds as a synthetic substrate for xeno-free expansion and differentiation of human induced pluripotent stem cells.

PubMed

Leong, Meng Fatt; Lu, Hong Fang; Lim, Tze Chiun; Du, Chan; Ma, Nina K L; Wan, Andrew C A

2016-12-01

The use of human induced pluripotent stem cells (hiPSCs) for clinical tissue engineering applications requires expansion and differentiation of the cells using defined, xeno-free substrates. The screening and selection of suitable synthetic substrates however, is tedious, as their performance relies on the inherent material properties. In the present work, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffolds were found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates which were shown to be pluripotent colonies. Immunostaining, PCR analyses, in vitro differentiation and in vivo teratoma formation studies demonstrated that these hiPSC aggregates could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency. Flow cytometry showed that more than 80% of the cell population stained positive for the pluripotent marker OCT4 at P1, P5 and P10. P10 cells could be differentiated to neuronal-like cells and cultured within the ESPS for up to 18months. Our results suggest the usefulness of a generic class of synthetic substrates, exemplified by ESPS, for 'trapped aggregate culture' of hiPSCs. To realize the potential of human induced pluripotent stem cells (hiPSCs) in clinical medicine, robust, xeno-free substrates for expansion and differentiation of iPSCs are required. In the existing literature, synthetic materials have been reported that meet the requirement for non-xenogeneic substrates. However, the self-renewal and differentiation characteristics of hiPSCs are affected differently by the biocompatibility and physico-chemical properties of individual substrates. Although some rules based on chemical structure and substrate rigidity have been developed, most of these efforts are still empirical, and most synthetic substrates must still be rigorously screened for suitability. In this paper, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffold was found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates. In the form of these trapped aggregates, we showed that hiPSCs could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency, following which they could be differentiated to a chosen lineage. We believe that this novel, generic class of synthetic substrates that employs 'trapped aggregate culture' for expansion and differentiation of hiPSCs is an important conceptual advance, and would be of high interest to the readership of Acta Biomaterialia. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2018-01-01

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

Generating size-controlled embryoid bodies using laser direct-write.

PubMed

Dias, A D; Unser, A M; Xie, Y; Chrisey, D B; Corr, D T

2014-06-01

Embryonic stem cells (ESCs) have the potential to self-renew and differentiate into any specialized cell type. One common method to differentiate ESCs in vitro is through embryoid bodies (EBs), three-dimensional cellular aggregates that spontaneously self-assemble and generally express markers for the three germ layers, endoderm, ectoderm, and mesoderm. It has been previously shown that both EB size and 2D colony size each influence differentiation. We hypothesized that we could control the size of the EB formed by mouse ESCs (mESCs) by using a cell printing method, laser direct-write (LDW), to control both the size of the initial printed colony and the local cell density in printed colonies. After printing mESCs at various printed colony sizes and printing densities, two-way ANOVAs indicated that the EB diameter was influenced by printing density after three days (p = 0.0002), while there was no effect of the printed colony diameter on the EB diameter at the same timepoint (p = 0.74). There was no significant interaction between these two factors. Tukey's honestly significant difference test showed that high-density colonies formed significantly larger EBs, suggesting that printed mESCs quickly aggregate with nearby cells. Thus, EBs can be engineered to a desired size by controlling printing density, which will influence the design of future differentiation studies. Herein, we highlight the capacity of LDW to control the local cell density and colony size independently, at prescribed spatial locations, potentially leading to better stem cell maintenance and directed differentiation.

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis

NASA Technical Reports Server (NTRS)

Nickerson, C. A.; Goodwin, T. J.; Terlonge, J.; Ott, C. M.; Buchanan, K. L.; Uicker, W. C.; Emami, K.; LeBlanc, C. L.; Ramamurthy, R.; Clarke, M. S.;

Three-Dimensional Tissue Assemblies: Novel Models for the Study of Salmonella enterica Serovar Typhimurium Pathogenesis

PubMed Central

Nickerson, Cheryl A.; Goodwin, Thomas J.; Terlonge, Jacqueline; Ott, C. Mark; Buchanan, Kent L.; Uicker, William C.; Emami, Kamal; LeBlanc, Carly L.; Ramamurthy, Rajee; Clarke, Mark S.; Vanderburg, Charles R.; Hammond, Timothy; Pierson, Duane L.

2001-01-01

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1α (IL-1α), IL-1β, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor β1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction. PMID:11598087

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Moriarity, D. M.; Campbell, P. S.

1993-01-01

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

In-silico analysis on biofabricating vascular networks using kinetic Monte Carlo simulations.

PubMed

Sun, Yi; Yang, Xiaofeng; Wang, Qi

2014-03-01

We present a computational modeling approach to study the fusion of multicellular aggregate systems in a novel scaffold-less biofabrication process, known as 'bioprinting'. In this novel technology, live multicellular aggregates are used as fundamental building blocks to make tissues or organs (collectively known as the bio-constructs,) via the layer-by-layer deposition technique or other methods; the printed bio-constructs embedded in maturogens, consisting of nutrient-rich bio-compatible hydrogels, are then placed in bioreactors to undergo the cellular aggregate fusion process to form the desired functional bio-structures. Our approach reported here is an agent-based modeling method, which uses the kinetic Monte Carlo (KMC) algorithm to evolve the cellular system on a lattice. In this method, the cells and the hydrogel media, in which cells are embedded, are coarse-grained to material's points on a three-dimensional (3D) lattice, where the cell-cell and cell-medium interactions are quantified by adhesion and cohesion energies. In a multicellular aggregate system with a fixed number of cells and fixed amount of hydrogel media, where the effect of cell differentiation, proliferation and death are tactically neglected, the interaction energy is primarily dictated by the interfacial energy between cell and cell as well as between cell and medium particles on the lattice, respectively, based on the differential adhesion hypothesis. By using the transition state theory to track the time evolution of the multicellular system while minimizing the interfacial energy, KMC is shown to be an efficient time-dependent simulation tool to study the evolution of the multicellular aggregate system. In this study, numerical experiments are presented to simulate fusion and cell sorting during the biofabrication process of vascular networks, in which the bio-constructs are fabricated via engineering designs. The results predict the feasibility of fabricating the vascular structures via the bioprinting technology and demonstrate the morphological development process during cellular aggregate fusion in various engineering designed structures. The study also reveals that cell sorting will perhaps not significantly impact the final fabricated products, should the maturation process be well-controlled in bioprinting.

Role of differential physical properties in emergent behavior of 3D cell co-cultures

NASA Astrophysics Data System (ADS)

Kolbman, Dan; Das, Moumita

2015-03-01

The biophysics of binary cell populations is of great interest in many biological processes, whether the formation of embryos or the initiation of tumors. During these processes, cells are surrounded by other cell types with different physical properties, often with important consequences. For example, recent experiments on a co-culture of breast cancer cells and healthy breast epithelial cells suggest that the mechanical mismatch between the two cell types may contribute to enhanced migration of the cancer cells. Here we explore how the differential physical properties of different cell types may influence cell-cell interaction, aggregation, and migration. To this end, we study a proof of concept model- a three-dimensional binary system of interacting, active, and deformable particles with different physical properties such as elastic stiffness, contractility, and particle-particle adhesion, using Langevin Dynamics simulations. Our results may provide insights into emergent behavior such as segregation and differential migration in cell co-cultures in three dimensions.

Surface modified alginate microcapsules for 3D cell culture

NASA Astrophysics Data System (ADS)

Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

2016-06-01

Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

Three-dimensional transgenic cell model to quantify genotoxic effects of space environment

NASA Astrophysics Data System (ADS)

Gonda, S. R.; Wu, H.; Pingerelli, P. L.; Glickman, B. W.

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of multiple copies of defined target genes for genotoxic assessment. Rat 2λ fibroblasts, genetically engineered to contain high-density target genes for mutagenesis (Stratagene, Inc., Austin, TX), were cocultured with human epithelial cells on Cytodex beads in the High Aspect Ratio Bioreactor (Synthecon, Inc, Houston, TX). Multi-bead aggregates were formed by day 5 following the complete covering of the beads by fibroblasts. Cellular retraction occurred 8-14 days after coculture initiation culminating in spheroids retaining few or no beads. Analysis of the resulting tissue assemblies revealed: multicellular spheroids, fibroblasts synthesized collagen, and cell viability was retained for the 30-day test period after removal from the bioreactor. Quantification of mutation at the LacI gene in Rat 2λ fibroblasts in spheroids exposed to 0-2 Gy neon using the Big Blue color assay (Stratagene, Inc.), revealed a linear dose-response for mutation induction. Limited sequencing analysis of mutant clones from 0.25 or 1 Gy exposures revealed a higher frequency of deletions and multiple base sequencing changes with increasing dose. These results suggest that the three-dimensional, multicellular tissue assembly model produced in NASA bioreactors are applicable to a wide variety of studies involving the quantification and identification of genotocity including measurement of the inherent damage incurred in Space.

Survey of Methods and Algorithms of Robot Swarm Aggregation

NASA Astrophysics Data System (ADS)

E Shlyakhov, N.; Vatamaniuk, I. V.; Ronzhin, A. L.

2017-01-01

The paper considers the problem of swarm aggregation of autonomous robots with the use of three methods based on the analogy of the behavior of biological objects. The algorithms substantiating the requirements for hardware realization of sensor, computer and network resources and propulsion devices are presented. Techniques for efficiency estimation of swarm aggregation via space-time characteristics are described. The developed model of the robot swarm reconfiguration into a predetermined three-dimensional shape is presented.

Production of Human Pluripotent Stem Cell Therapeutics Under Defined Xeno-free Conditions: Progress and Challenges

PubMed Central

Fan, Yongjia; Wu, Jincheng; Ashok, Preeti; Hsiung, Michael; Tzanakakis, Emmanuel S.

2014-01-01

Recent advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the realization of their clinical potential. Nonetheless, tissue engineering and regenerative medicine applications will require the generation of hPSC products well beyond the laboratory scale. This also mandates the production of hPSC therapeutics in fully-defined, xeno-free systems and in a reproducible manner. Toward this goal, we summarize current developments in defined media free of animal-derived components for hPSC culture. Bioinspired and synthetic extracellular matrices for the attachment growth and differentiation of hPSCs are also reviewed. Given that most progress in xeno-free medium and substrate development has been demonstrated in two-dimensional rather than three dimensional culture systems, translation from the former to the latter poses unique difficulties. These challenges are discussed in the context of cultivation platforms of hPSCs as aggregates, on microcarriers or after encapsulation in biocompatible scaffolds. PMID:25077810

Production of human pluripotent stem cell therapeutics under defined xeno-free conditions: progress and challenges.

PubMed

Fan, Yongjia; Wu, Jincheng; Ashok, Preeti; Hsiung, Michael; Tzanakakis, Emmanuel S

2015-02-01

Recent advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the realization of their clinical potential. Nonetheless, tissue engineering and regenerative medicine applications will require the generation of hPSC products well beyond the laboratory scale. This also mandates the production of hPSC therapeutics in fully-defined, xeno-free systems and in a reproducible manner. Toward this goal, we summarize current developments in defined media free of animal-derived components for hPSC culture. Bioinspired and synthetic extracellular matrices for the attachment, growth and differentiation of hPSCs are also reviewed. Given that most progress in xeno-free medium and substrate development has been demonstrated in two-dimensional rather than three dimensional culture systems, translation from the former to the latter poses unique difficulties. These challenges are discussed in the context of cultivation platforms of hPSCs as aggregates, on microcarriers or after encapsulation in biocompatible scaffolds.

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Construction of bioengineered hepatic tissue derived from human umbilical cord mesenchymal stem cells via aggregation culture in porcine decellularized liver scaffolds.

PubMed

Li, Yi; Wu, Qiong; Wang, Yujia; Li, Li; Chen, Fei; Shi, Yujun; Bao, Ji; Bu, Hong

2017-01-01

An individualized, tissue-engineered liver suitable for transplanting into a patient with liver disease would be of great benefit to the patient and the healthcare system. The tissue-engineered liver would possess the functions of the original healthy organ. Two fields of study, (i) using decellularized tissue as cell scaffolding, and (ii) stem cell differentiation into functional cells, are coming together to make this concept feasible. The decellularized liver scaffolds (DLS) can interact with cells to promote cell differentiation and signal transduction and three-dimensional (3D) stem cell aggregations can maintain the phenotypes and improve functions of stem cells after differentiation by undergoing cell-cell contact. Although the effects of DLS and stem cell aggregation culture have been intensively studied, few observations about the interaction between the two have been achieved. We established a method that combines the use of decellularized liver scaffolds and aggregation culture of MSCs (3D-DLS) and explored the effects of the two on hepatic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in bioengineered hepatic tissue. A higher percentage of albumin-producing cells, higher levels of liver-specific transcripts, higher urea cycle-related transcripts, and lower levels of stem cell-specific transcripts were observed in the 3D-DLS group when compared to that of hUC-MSCs in monolayer culture (2D), aggregation culture (3D), monolayer on DLS culture (2D-DLS). The gene arrays also indicated that 3D-DLS induced the differentiation from the hUC-MSC phenotype to the PHH phenotype. Liver-specific proteins albumin, CK-18, and glycogen storage were highly positive in the 3D-DLS group. Albumin secretion and ammonia conversion to urea were more effective with a higher cell survival rate in the 3D-DLS group for 14 days. This DLS and aggregation combination culture system provides a novel method to improve hepatic differentiation, maintain phenotype of hepatocyte-like cells and sustain survival for 14 days in vitro. This is a promising strategy to use to construct bioengineered hepatic tissue. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment.

PubMed

Reyes, Darwin R; Hong, Jennifer S; Elliott, John T; Gaitan, Michael

2011-08-16

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.

Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids.

PubMed

Schmitz, Alexander; Fischer, Sabine C; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H K

2017-03-03

Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid's size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 10 5 to 1 × 10 6  cells/mm 3 . Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.

Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon.

PubMed

Duangsuwan, Pornsawan; Phoungpetchara, Ittipon; Tinikul, Yotsawan; Poljaroen, Jaruwan; Wanichanon, Chaitip; Sobhon, Prasert

2008-04-01

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.

Phase separation like dynamics during Myxococcus xanthus fruiting body formation

NASA Astrophysics Data System (ADS)

Liu, Guannan; Thutupalli, Shashi; Wigbers, Manon; Shaevitz, Joshua

2015-03-01

Collective motion exists in many living organisms as an advantageous strategy to help the entire group with predation, forage, and survival. However, the principles of self-organization underlying such collective motions remain unclear. During various developmental stages of the soil-dwelling bacterium, Myxococcus xanthus, different types of collective motions are observed. In particular, when starved, M. xanthus cells eventually aggregate together to form 3-dimensional structures (fruiting bodies), inside which cells sporulate in response to the stress. We study the fruiting body formation process as an out of equilibrium phase separation process. As local cell density increases, the dynamics of the aggregation M. xanthus cells switch from a spatio-temporally random process, resembling nucleation and growth, to an emergent pattern formation process similar to a spinodal decomposition. By employing high-resolution microscopy and a video analysis system, we are able to track the motion of single cells within motile collective groups, while separately tuning local cell density, cell velocity and reversal frequency, probing the multi-dimensional phase space of M. xanthus development.

Generation of multicellular tumor spheroids by the hanging-drop method.

PubMed

Timmins, Nicholas E; Nielsen, Lars K

2007-01-01

Owing to their in vivo-like characteristics, three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are gaining increasing popularity as an in vitro model of tumors. A straightforward and simple approach to the cultivation of these MCTS is the hanging-drop method. Cells are suspended in droplets of medium, where they develop into coherent 3D aggregates and are readily accessed for analysis. In addition to being simple, the method eliminates surface interactions with an underlying substratum (e.g., polystyrene plastic or agarose), requires only a low number of starting cells, and is highly reproducible. This method has also been applied to the co-cultivation of mixed cell populations, including the co-cultivation of endothelial cells and tumor cells as a model of early tumor angiogenesis.

Reassembly of Anterior Pituitary Organization by Hanging Drop Three-Dimensional Cell Culture

PubMed Central

Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components—collagens and laminin—were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization. PMID:24023396

Reassembly of anterior pituitary organization by hanging drop three-dimensional cell culture.

PubMed

Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2013-08-29

The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.

Three-dimensional forward modeling of DC resistivity using the aggregation-based algebraic multigrid method

NASA Astrophysics Data System (ADS)

Chen, Hui; Deng, Ju-Zhi; Yin, Min; Yin, Chang-Chun; Tang, Wen-Wu

2017-03-01

To speed up three-dimensional (3D) DC resistivity modeling, we present a new multigrid method, the aggregation-based algebraic multigrid method (AGMG). We first discretize the differential equation of the secondary potential field with mixed boundary conditions by using a seven-point finite-difference method to obtain a large sparse system of linear equations. Then, we introduce the theory behind the pairwise aggregation algorithms for AGMG and use the conjugate-gradient method with the V-cycle AGMG preconditioner (AGMG-CG) to solve the linear equations. We use typical geoelectrical models to test the proposed AGMG-CG method and compare the results with analytical solutions and the 3DDCXH algorithm for 3D DC modeling (3DDCXH). In addition, we apply the AGMG-CG method to different grid sizes and geoelectrical models and compare it to different iterative methods, such as ILU-BICGSTAB, ILU-GCR, and SSOR-CG. The AGMG-CG method yields nearly linearly decreasing errors, whereas the number of iterations increases slowly with increasing grid size. The AGMG-CG method is precise and converges fast, and thus can improve the computational efficiency in forward modeling of three-dimensional DC resistivity.

Comparison of three optical methods to study erythrocyte aggregation.

PubMed

Zhao, H; Wang, X; Stoltz, J F

1999-01-01

The aim of this work was to evaluate three optical methods designed to determine erythrocyte aggregation: Erythroaggregometer (EA; Regulest, France), Laser-assisted Optical Rotational Cell Analyzer (LORCA; Mechatronics, Netherlands) and Fully Automatic Erythrocyte Aggregometer (FAEA; Myrenne, GmbH, Germany). Blood samples were taken from fifty donors (26 males and 24 females). The aggregation of normal red blood cell (RBC) and RBCs suspended in three normo- and hyperaggregating suspending media was studied. The results revealed some significant correlations between parameters measured by these instruments, in particular, between the indexes of aggregation of EA and LORCA. Further, RBC aggregation of multiple myeloma patients was also studied and a hyper erythrocyte aggregation state was found by EA and LORCA.

3D simulations of early blood vessel formation

NASA Astrophysics Data System (ADS)

Cavalli, F.; Gamba, A.; Naldi, G.; Semplice, M.; Valdembri, D.; Serini, G.

2007-08-01

Blood vessel networks form by spontaneous aggregation of individual cells migrating toward vascularization sites (vasculogenesis). A successful theoretical model of two-dimensional experimental vasculogenesis has been recently proposed, showing the relevance of percolation concepts and of cell cross-talk (chemotactic autocrine loop) to the understanding of this self-aggregation process. Here we study the natural 3D extension of the computational model proposed earlier, which is relevant for the investigation of the genuinely three-dimensional process of vasculogenesis in vertebrate embryos. The computational model is based on a multidimensional Burgers equation coupled with a reaction diffusion equation for a chemotactic factor and a mass conservation law. The numerical approximation of the computational model is obtained by high order relaxed schemes. Space and time discretization are performed by using TVD schemes and, respectively, IMEX schemes. Due to the computational costs of realistic simulations, we have implemented the numerical algorithm on a cluster for parallel computation. Starting from initial conditions mimicking the experimentally observed ones, numerical simulations produce network-like structures qualitatively similar to those observed in the early stages of in vivo vasculogenesis. We develop the computation of critical percolative indices as a robust measure of the network geometry as a first step towards the comparison of computational and experimental data.

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids.

PubMed

Bartnik, E; Sievers, A

1988-11-01

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 μm and is positioned in the center of the apical Golgi-vesicle accumulation ("Spitzenkörper"). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.

Growing Three-Dimensional Corneal Tissue in a Bioreactor

NASA Technical Reports Server (NTRS)

Spaulding, Glen F.; Goodwin, Thomas J.; Aten, Laurie; Prewett, Tacey; Fitzgerald, Wendy S.; OConnor, Kim; Caldwell, Delmar; Francis, Karen M.

2003-01-01

Spheroids of corneal tissue about 5 mm in diameter have been grown in a bioreactor from an in vitro culture of primary rabbit corneal cells to illustrate the production of optic cells from aggregates and tissue. In comparison with corneal tissues previously grown in vitro by other techniques, this tissue approximates intact corneal tissue more closely in both size and structure. This novel three-dimensional tissue can be used to model cell structures and functions in normal and abnormal corneas. Efforts continue to refine the present in vitro method into one for producing human corneal tissue to overcome the chronic shortage of donors for corneal transplants: The method would be used to prepare corneal tissues, either from in vitro cultures of a patient s own cells or from a well-defined culture from another human donor known to be healthy. As explained in several articles in prior issues of NASA Tech Briefs, generally cylindrical horizontal rotating bioreactors have been developed to provide nutrient-solution environments conducive to the 30 NASA Tech Briefs, October 2003 growth of delicate animal cells, with gentle, low-shear flow conditions that keep the cells in suspension without damaging them. The horizontal rotating bioreactor used in this method, denoted by the acronym "HARV," was described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), NASA Tech Briefs, Vol. 16, No. 5 (May, 1992), page 150.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

PubMed

Yamamoto, Mioko; Kawashima, Nobuyuki; Takashino, Nami; Koizumi, Yu; Takimoto, Koyo; Suzuki, Noriyuki; Saito, Masahiro; Suda, Hideaki

2014-03-01

Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Three-Dimensional Analytical Modeling of Diffusion-Limited Solute Transport.

DTIC Science & Technology

1986-07-01

displace- ment through an aggregated oxisol , Soil Sci. Soc. Am. J., 46, 471-476. Nkedi-Kizza, P., J. W. Biggar, H. M. Selim, M. Th. van Genuchten, P. J...aggregated oxisol , Water Resources Research, 20(8), 1123-1130. National Research Council (NRC), 1984. Groundwater Contamination, Panel on Groundwater

Multiscale modelling of hydraulic conductivity in vuggy porous media

PubMed Central

Daly, K. R.; Roose, T.

2014-01-01

Flow in both saturated and non-saturated vuggy porous media, i.e. soil, is inherently multiscale. The complex microporous structure of the soil aggregates and the wider vugs provides a multitude of flow pathways and has received significant attention from the X-ray computed tomography (CT) community with a constant drive to image at higher resolution. Using multiscale homogenization, we derive averaged equations to study the effects of the microscale structure on the macroscopic flow. The averaged model captures the underlying geometry through a series of cell problems and is verified through direct comparison to numerical simulations of the full structure. These methods offer significant reductions in computation time and allow us to perform three-dimensional calculations with complex geometries on a desktop PC. The results show that the surface roughness of the aggregate has a significantly greater effect on the flow than the microstructure within the aggregate. Hence, this is the region in which the resolution of X-ray CT for image-based modelling has the greatest impact. PMID:24511248

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

PubMed

Kino-Oka, Masahiro; Maeda, Yoshikatsu; Yamamoto, Takeyuki; Sugawara, Katsura; Taya, Masahito

2005-03-01

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

Microstructured block copolymer surfaces for control of microbe capture and aggregation

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

Hansen, Ryan R; Shubert, Katherine R; Morrell, Jennifer L.

2014-01-01

The capture and arrangement of surface-associated microbes is influenced by biochemical and physical properties of the substrate. In this report, we develop lectin-functionalized substrates containing patterned, three-dimensional polymeric structures of varied shapes and densities and use these to investigate the effects of topology and spatial confinement on lectin-mediated microbe capture. Films of poly(glycidyl methacrylate)-block-4,4-dimethyl-2-vinylazlactone (PGMA-b-PVDMA) were patterned on silicon surfaces into line or square grid patterns with 5 m wide features and varied edge spacing. The patterned films had three-dimensional geometries with 900 nm film thickness. After surface functionalization with wheat germ agglutinin, the size of Pseudomonas fluorescens aggregates capturedmore » was dependent on the pattern dimensions. Line patterns with edge spacing of 5 m or less led to the capture of individual microbes with minimal formation of aggregates, while grid patterns with the same spacing also captured individual microbes with further reduction in aggregation. Both geometries allowed for increases in aggregate size distribution with increased in edge spacing. These engineered surfaces combine spatial confinement with affinity-based microbe capture based on exopolysaccharide content to control the degree of microbe aggregation, and can also be used as a platform to investigate intercellular interactions and biofilm formation in microbial populations of controlled sizes.« less

Progress in Integrative Biomaterial Systems to Approach Three-Dimensional Cell Mechanotransduction

PubMed Central

Zhang, Ying; Liao, Kin; Li, Chuan; Lai, Alvin C.K.; Foo, Ji-Jinn

2017-01-01

Mechanotransduction between cells and the extracellular matrix regulates major cellular functions in physiological and pathological situations. The effect of mechanical cues on biochemical signaling triggered by cell–matrix and cell–cell interactions on model biomimetic surfaces has been extensively investigated by a combination of fabrication, biophysical, and biological methods. To simulate the in vivo physiological microenvironment in vitro, three dimensional (3D) microstructures with tailored bio-functionality have been fabricated on substrates of various materials. However, less attention has been paid to the design of 3D biomaterial systems with geometric variances, such as the possession of precise micro-features and/or bio-sensing elements for probing the mechanical responses of cells to the external microenvironment. Such precisely engineered 3D model experimental platforms pave the way for studying the mechanotransduction of multicellular aggregates under controlled geometric and mechanical parameters. Concurrently with the progress in 3D biomaterial fabrication, cell traction force microscopy (CTFM) developed in the field of cell biophysics has emerged as a highly sensitive technique for probing the mechanical stresses exerted by cells onto the opposing deformable surface. In the current work, we first review the recent advances in the fabrication of 3D micropatterned biomaterials which enable the seamless integration with experimental cell mechanics in a controlled 3D microenvironment. Then, we discuss the role of collective cell–cell interactions in the mechanotransduction of engineered tissue equivalents determined by such integrative biomaterial systems under simulated physiological conditions. PMID:28952551

Unique cell culture systems for ground based research

NASA Technical Reports Server (NTRS)

Lewis, Marian L.

1990-01-01

The horizontally rotating fluid-filled, membrane oxygenated bioreactors developed at NASA Johnson for spacecraft applications provide a powerful tool for ground-based research. Three-dimensional aggregates formed by cells cultured on microcarrier beads are useful for study of cell-cell interactions and tissue development. By comparing electron micrographs of plant seedlings germinated during Shuttle flight 61-C and in an earth-based rotating bioreactor it is shown that some effects of microgravity are mimicked. Bioreactors used in the UAH Bioreactor Laboratory will make it possible to determine some of the effects of altered gravity at the cellular level. Bioreactors can be valuable for performing critical, preliminary-to-spaceflight experiments as well as medical investigations such as in vitro tumor cell growth and chemotherapeutic drug response; the enrichment of stem cells from bone marrow; and the effect of altered gravity on bone and muscle cell growth and function and immune response depression.

Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth

PubMed Central

Luef, Birgit; Fakra, Sirine C; Csencsits, Roseann; Wrighton, Kelly C; Williams, Kenneth H; Wilkins, Michael J; Downing, Kenneth H; Long, Philip E; Comolli, Luis R; Banfield, Jillian F

2013-01-01

Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L2,3 absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)–Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension. PMID:23038172

Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth.

PubMed

Luef, Birgit; Fakra, Sirine C; Csencsits, Roseann; Wrighton, Kelly C; Williams, Kenneth H; Wilkins, Michael J; Downing, Kenneth H; Long, Philip E; Comolli, Luis R; Banfield, Jillian F

2013-02-01

Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L(2,3) absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)-Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.

Three-dimensional polymer scaffolds for enhanced differentiation of human mesenchymal stem cells to hepatocyte-like cells: a comparative study.

PubMed

Chitrangi, Swati; Nair, Prabha; Khanna, Aparna

2017-08-01

Stem cell-based tissue engineering has emerged as a promising avenue for the treatment of liver diseases and as drug metabolism and toxicity models in drug discovery and development. The in vitro simulation of a micro-environmental niche for hepatic differentiation remains elusive, due to lack of information about crucial factors for the stem cell niche. For generation of functional hepatocytes, an in vivo three-dimensional (3D) micro-environment and architecture should be reproduced. Towards this, we fabricated three scaffolds as dextran-gelatin (DG1), chitosan-hyaluronic acid (CH1) and gelatin-vinyl acetate (GEVAC). Hepatic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was induced by culturing hUC-MSCs on these scaffolds. The scaffolds support hepatic differentiation by mimicking the native extracellular matrix (ECM) micro-environment and architecture to facilitate 3D cell-cell and cell-matrix interactions. The expression of hepatic markers, glycogen storage, urea production, albumin secretion and cytochrome P450 (CYP450) activity indicated the hepatic differentiation of hUC-MSCs. The differentiated hUC-MSCs on the 3D scaffolds formed hepatospheroids (3D hepatocyte aggregates), as illustrated by scanning electron microscopy (SEM), confocal microscopy and cytoskeleton organization. It was observed that the 3D scaffolds supported improved cell morphology, expression of hepatic markers and metabolic activities, as compared to Matrigel-coated plates. To the best of our knowledge, this is the first report demonstrating the use of a well-characterized scaffold (GEVAC) for enhanced differentiation of hUC-MSCs to hepatocyte-like cells (HLCs). Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Three-photon-excited luminescence from unsymmetrical cyanostilbene aggregates: morphology tuning and targeted bioimaging.

PubMed

Mandal, Amal Kumar; Sreejith, Sivaramapanicker; He, Tingchao; Maji, Swarup Kumar; Wang, Xiao-Jun; Ong, Shi Li; Joseph, James; Sun, Handong; Zhao, Yanli

2015-05-26

We report an experimental observation of aggregation-induced enhanced luminescence upon three-photon excitation in aggregates formed from a class of unsymmetrical cyanostilbene derivatives. Changing side chains (-CH3, -C6H13, -C7H15O3, and folic acid) attached to the cyanostilbene core leads to instantaneous formation of aggregates with sizes ranging from micrometer to nanometer scale in aqueous conditions. The crystal structure of a derivative with a methyl side chain reveals the planarization in the unsymmetrical cyanostilbene core, causing luminescence from corresponding aggregates upon three-photon excitation. Furthermore, folic acid attached cyanostilbene forms well-dispersed spherical nanoaggregates that show a high three-photon cross-section of 6.0 × 10(-80) cm(6) s(2) photon(-2) and high luminescence quantum yield in water. In order to demonstrate the targeted bioimaging capability of the nanoaggregates, three cell lines (HEK293 healthy cell line, MCF7 cancerous cell line, and HeLa cancerous cell line) were employed for the investigations on the basis of their different folate receptor expression level. Two kinds of nanoaggregates with and without the folic acid targeting ligand were chosen for three-photon bioimaging studies. The cell viability of three types of cells incubated with high concentration of nanoaggregates still remained above 70% after 24 h. It was observed that the nanoaggregates without the folic acid unit could not undergo the endocytosis by both healthy and cancerous cell lines. No obvious endocytosis of folic acid attached nanoaggregates was observed from the HEK293 and MCF7 cell lines having a low expression of the folate receptor. Interestingly, a significant amount of endocytosis and internalization of folic acid attached nanoaggregates was observed from HeLa cells with a high expression of the folate receptor under three-photon excitation, indicating targeted bioimaging of folic acid attached nanoaggregates to the cancer cell line. This study presents a paradigm of using organic nanoaggregates for targeted three-photon bioimaging.

Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

PubMed

Christakou, Athanasia E; Ohlin, Mathias; Önfelt, Björn; Wiklund, Martin

2015-08-07

We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

NASA Astrophysics Data System (ADS)

Berdeu, Anthony; Laperrousaz, Bastien; Bordy, Thomas; Morales, S.; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

2018-02-01

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as 5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

PubMed

Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Analysis and comparison of oxygen consumption of HepG2 cells in a monolayer and three-dimensional high density cell culture by use of a matrigrid®.

PubMed

Weise, Frank; Fernekorn, Uta; Hampl, Jörg; Klett, Maren; Schober, Andreas

2013-09-01

By the use of a MatriGrid® we have established a three-dimensional high density cell culture. The MatriGrid® is a culture medium permeable, polymeric scaffold with 187 microcavities. In these cavities (300 μm diameter and 207 μm deep) the cells can growth three-dimensionally. For these experiments we measured the oxygen consumption of HepG2 cell cultures in order to optimize cultivation conditions. We measured and compared the oxygen consumption, growth rate and vitality under three different cultivation conditions: monolayer, three-dimensional static and three-dimensional actively perfused. The results show that the cells in a three-dimensional cell culture consume less oxygen as in a monolayer cell culture and that the actively perfused three-dimensional cell culture in the MatriGrid® has a similar growth rate and vitality as the monolayer culture. Copyright © 2013 Wiley Periodicals, Inc.

Dissipative particle dynamics simulations of deformation and aggregation of healthy and diseased red blood cells in a tube flow

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

Ye, Ting; Phan-Thien, Nhan, E-mail: Nhan@nus.edu.sg; Khoo, Boo Cheong

In this paper, we report simulation results assessing the deformation and aggregation of mixed healthy and malaria-infected red blood cells (RBCs) in a tube flow. A three dimensional particle model based on Dissipative Particle Dynamics (DPD) is developed to predict the tube flow containing interacting cells. The cells are also modelled by DPD, with a Morse potential to characterize the cell-cell interaction. As validation tests, a single RBC in a tube flow and two RBCs in a static flow are simulated to examine the cell deformation and intercellular interaction, respectively. The study of two cells, one healthy and the othermore » malaria-infected RBCs in a tube flow demonstrates that the malaria-infected RBC (in the leading position along flow direction) has different effects on the healthy RBC (in the trailing position) at the different stage of parasite development or at the different capillary number. With parasitic development, the malaria-infected RBC gradually loses its deformability, and in turn the corresponding trailing healthy RBC also deforms less due to the intercellular interaction. With increasing capillary number, both the healthy and malaria-infected RBCs are likely to undergo an axisymmetric motion. The minimum intercellular distance becomes small enough so that rouleaux is easily formed, i.e., the healthy and malaria-infected RBCs are difficultly disaggregated.« less

Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells.

PubMed

Koehler, Karl R; Nie, Jing; Longworth-Mills, Emma; Liu, Xiao-Ping; Lee, Jiyoon; Holt, Jeffrey R; Hashino, Eri

2017-06-01

The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR-Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.

Aeromonas species exhibit aggregative adherence to HEp-2 cells.

PubMed Central

Neves, M S; Nunes, M P; Milhomem, A M

1994-01-01

Clinical and environmental isolates of Aeromonas species (five A. hydrophila isolates, three A. caviae isolates, and two A. sobria isolates) were tested for their adherence to HEp-2 cells. Clinical isolates of A. hydrophila and A. sobria exhibited aggregative adherence similar to that presented by enteroadherent-aggregative Escherichia coli. Bacterial aggregates adhered to cells with a typical "stacked-brick" appearance. In contrast, A. caviae strains showed a diffuse adherence pattern. Images PMID:8027331

Characterization of mesenchymal stem cells and fibrochondrocytes in three-dimensional co-culture: analysis of cell shape, matrix production, and mechanical performance.

PubMed

McCorry, Mary Clare; Puetzer, Jennifer L; Bonassar, Lawrence J

2016-03-12

Bone marrow mesenchymal stem cells (MSCs) have shown positive therapeutic effects for meniscus regeneration and repair. Preliminary in vitro work has indicated positive results for MSC applications for meniscus tissue engineering; however, more information is needed on how to direct MSC behavior. The objective of this study was to examine the effect of MSC co-culture with primary meniscal fibrochondrocytes (FCCs) in a three-dimensional collagen scaffold in fibrochondrogenic media. Co-culture of MSCs and FCCs was hypothesized to facilitate the transition of MSCs to a FCC cell phenotype as measured by matrix secretion and morphology. MSCs and FCCs were isolated from bovine bone marrow and meniscus, respectively. Cells were seeded in a 20 mg/mL high-density type I collagen gel at MSC:FCC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs were cultured for up to 2 weeks and then analyzed for cell morphology, glycosaminoglycan content, collagen content, and production of collagen type I, II, and X. Cells were homogeneously mixed throughout the scaffold and cells had limited direct cell-cell contact. After 2 weeks in culture, MSCs transitioned from a spindle-like morphology toward a rounded phenotype, while FCCs remained rounded throughout culture. Although MSC shape changed with culture, the overall size was significantly larger than FCCs throughout culture. While 75:25 and 100:0 (MSC mono-culture) culture groups produced significantly more glycosaminoglycan (GAG)/DNA than FCCs in mono-culture, GAG retention was highest in 50:50 co-cultures. Similarly, the aggregate modulus was highest in 100:0 and 50:50 co-cultures. All samples contained both collagen types I and II after 2 weeks, and collagen type X expression was evident only in MSC mono-culture gels. MSCs shift to a FCC morphology in both mono- and co-culture. Co-culture reduced hypertrophy by MSCs, indicated by collagen type X. This study shows that MSC phenotype can be influenced by indirect homogeneous cell culture in a three-dimensional gel, demonstrating the applicability of MSCs in meniscus tissue engineering applications.

High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array.

PubMed

Tung, Yi-Chung; Hsiao, Amy Y; Allen, Steven G; Torisawa, Yu-suke; Ho, Mitchell; Takayama, Shuichi

2011-02-07

Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.

The effects of an RGD-PAMAM dendrimer conjugate in 3D spheroid culture on cell proliferation, expression and aggregation.

PubMed

Jiang, Li-Yang; Lv, Bing; Luo, Ying

2013-04-01

By presenting biomolecular ligands on the surface in high density, ligand-decorated dendrimers are capable of binding to membrane receptors and cells with specificity and avidity. Despite the various uses, fundamental investigations on ligand-dendrimer conjugates have mainly focused on their binding behavior with cells, whereas their potential bioactivity and applications in multicellular systems, especially in three-dimensional (3D) culture systems, remains untapped. In this study, a typical adhesive peptide ligand - RGD - was modified to generation 4 polyamidoamine (PAMAM), and the bioactivity of suspended RGD-PAMAM conjugates was investigated on cells cultured as multicellular spheroids. Our results demonstrate that the RGD-PAMAM conjugates, after being incorporated into the 3D spheroids, were able to promote cellular proliferation and aggregation, and affect the mRNA expression of extracellular factors by NIH 3T3 cells. These bioactive functions were multivalency-dependent, as none of similar effects was observed for monovalent RGD ligand. Our study suggests that multivalent ligand-dendrimer conjugates may act as a unique type of artificial factors to mediate the cellular microenvironment in 3D culture, a property attributable to the spatial organization of the ligands and possible "cell-gluing" function of multivalent conjugates. This new finding opens the door for further exploring multivalent ligand-dendrimer conjugates for applications in 3D cell culture and tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

Neuroglian is expressed on cells destined to form the prothoracic glands of Manduca embryos as they segregate from surrounding cells and rearrange during morphogenesis.

PubMed

Chen, C L; Lampe, D J; Robertson, H M; Nardi, J B

1997-01-01

A cell surface protein (3B11) is differentially expressed in the embryonic labial segment of Manduca as two circular monolayers of epithelial cells invaginate and segregate from surrounding epithelial cells. The cells that invaginate and preferentially express 3B11 represent the presumptive prothoracic glands. These cells continue to express protein 3B11 as they rearrange to form first a three-dimensional aggregate and later anastomosing filaments of cells. In the differentiated prothoracic gland, expression of 3B11 is restricted to sites of cell-cell contact. Cloning and sequencing of the cDNA for protein 3B11 revealed that this protein is the Manduca counterpart of Drosophila neuroglian and mouse L1. These surface proteins are known to function as adhesion/recognition molecules during development. Manduca neuroglian shares 58 and 31% identity respectively with the Drosophila and mouse proteins and has a cytoplasmic domain of over 100 amino acids.

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

PubMed

Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

2016-05-01

Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P < 0.0001 and P = 0.0078, respectively. There were no statistically significant differences in the numbers of telomere aggregates or the nuclear volumes between the tumor and normal epithelial cells. Secondary analyses examined the effects of age on 3D telomere architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture between the ESCC and normal epithelial tissues. However, there were no differences observed between the young and old patients. © 2015 International Society for Diseases of the Esophagus.

Ethanol-dispersed and antibody-conjugated polymer nanofibers for the selective capture and 3-dimensional culture of EpCAM-positive cells.

PubMed

Yoon, Junghyo; Yoon, Hee-Sook; Shin, Yoojin; Kim, Sanghyun; Ju, Youngjun; Kim, Jungbae; Chung, Seok

2017-07-01

Electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (NFs) were used as a platform for the selective capture and three-dimensional culture of EpCAM-positive cells in cell culture medium and whole blood. The NFs were treated with streptavidin to facilitate bond formation between the amino groups of streptavidin and the maleic anhydride groups of the NFs. A biotinylated anti-EpCAM monoclonal antibody (mAb) was attached to the streptavidin-conjugated NFs via the selective binding of streptavidin and biotin. Upon simple mixing and shaking with EpCAM-positive cancer cells in a wide concentration range from 10 to 1000,000 cells per 10mL, the mAb-attached NFs (mAb-NFs) captured the Ep-CAM positive cells in an efficiency of 59%-67% depending on initial cell concentrations, with minor mechanical capture of 14%-36%. Captured cells were directly cultured, forming cell aggregates, in the NF matrix, which ensures the cell proliferation and follow-up analysis. Furthermore, the capture capacity of mAb-NFs was assessed in the presence of whole blood and blood lysates, indicating cluster formation that captured target cells. It is anticipated that the antibody-attached NFs can be employed for the capture and analysis of very rare EpCAM positive circulating cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Preparation and Characterization of Monomodal Grapevine Virus A Capsid Protein.

PubMed

Santana, Vinícius S; Mariutti, Ricardo B; Eberle, Raphael J; Ullah, Anwar; Caruso, Icaro P; Arni, Raghuvir K

2015-01-01

Grapevine virus A (GVA), a flexible filament of approximately 800 nm in length is composed of capsid subunits that spontaneously assembles around a positive sense genomic RNA. In addition to encapsidation, plant viruses capsid proteins (CPs) participate in other processes throughout infection and GVA CP is involved in cell-to-cell translocation of the virus. A protocol was developed to obtain low-molecular weight GVA-CP that is not prone to aggregation and spontaneous assembly and this was characterized by circular dichroism and dynamic light scattering. These results indicate the suitably of GVA-CP for X-ray crystallographic and NMR studies that should lead to the elucidation of the first three-dimensional structure of a flexible filamentous virus from the Betaflexiviridae family.

Three-Dimensional Conductive Nanocomposites Based on Multiwalled Carbon Nanotube Networks and PEDOT:PSS as a Flexible Transparent Electrode for Optoelectronics.

PubMed

Cho, Er-Chieh; Li, Chiu-Ping; Huang, Jui-Hsiung; Lee, Kuen-Chan; Huang, Jen-Hsien

2015-06-03

We have synthesized conductive nanocomposites composed of multiwalled carbon nanotubes (MWCNTs) and Au nanoparticles (NPs). The Au NPs with an average size of approximately 4.3 nm are uniformly anchored on the MWCNT. After being exposed to microwave (MW) plasma irradiation, the anchored Au NPs melt and fuse, leading to larger aggregates (34 nm) that can connect the MWCNT forming a three-dimensional conducting network. The formation of a continuous MWCNT network can produce more a conductive pathway, leading to lower sheet resistance. When the Au-MWCNT is dispersed in the highly conductive polymer, poly(ethylene dioxythiophene):polystyrenesulfonate ( PSS), we can obtain solution-processable composite formulations for the preparation of a flexible transparent electrode. The resulting Au-MWCNT/PEDOT:PSS hybrid films possess a sheet resistance of 51 Ω/sq with a transmittance of 86.2% at 550 nm. We also fabricate flexible organic solar cells and electrochromic devices to demonstrate the potential use of the as-prepared composite electrodes. Compared with the indium tin oxide-based devices, both the solar cells and electrochromic devices with the composites incorporated as a transparent electrode deliver comparable performance.

Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-09-01

Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

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 cell culture. Copyright © 2015 Elsevier B.V. All rights reserved.

Acoustic Coherent Backscatter Enhancement from Aggregations of Point Scatterers

DTIC Science & Technology

2015-09-30

and far-field acoustic multiple scattering from two- and now three-dimensional aggregations of omnidirectional point scatterers to determine the...an aggregation of omnidirectional point scatterers [1]. If ψ(r) is the harmonic acoustic pressure field at frequency ω at the point r and ψ0(r) is... scattered field and is given by the sum in (1), N is the number of scatterers , gn is the scattering coefficient of the nth scatterer , ψn(rn) is the field

Tissue Engineering Under Microgravity Conditions-Use of Stem Cells and Specialized Cells.

PubMed

Grimm, Daniela; Egli, Marcel; Krüger, Marcus; Riwaldt, Stefan; Corydon, Thomas J; Kopp, Sascha; Wehland, Markus; Wise, Petra; Infanger, Manfred; Mann, Vivek; Sundaresan, Alamelu

2018-03-29

Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine. This review will focus on the current knowledge of the use of stem cells and specialized cells for tissue engineering under simulated microgravity conditions. We will report on recent advancements in the ability to construct 3D aggregates from various cell types using devices originally created to prepare for spaceflights such as the random positioning machine (RPM), the clinostat, or the NASA-developed rotating wall vessel (RWV) bioreactor, to engineer various tissues such as preliminary vessels, eye tissue, bone, cartilage, multicellular cancer spheroids, and others from different cells. In addition, stem cells had been investigated under microgravity for the purpose to engineer adipose tissue, cartilage, or bone. Recent publications have discussed different changes of stem cells when exposed to microgravity and the relevant pathways involved in these biological processes. Tissue engineering in microgravity is a new technique to produce organoids, spheroids, or tissues with and without scaffolds. These 3D aggregates can be used for drug testing studies or for coculture models. Multicellular tumor spheroids may be interesting for radiation experiments in the future and to reduce the need for in vivo experiments. Current achievements using cells from patients engineered on the RWV or on the RPM represent an important step in the advancement of techniques that may be applied in translational Regenerative Medicine.

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration.

PubMed

Van de Walle, Aurore; Wilhelm, Claire; Luciani, Nathalie

2017-04-27

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Squaramide-Based Supramolecular Materials for Three-Dimensional Cell Culture of Human Induced Pluripotent Stem Cells and Their Derivatives

PubMed Central

2018-01-01

Synthetic hydrogel materials can recapitulate the natural cell microenvironment; however, it is equally necessary that the gels maintain cell viability and phenotype while permitting reisolation without stress, especially for use in the stem cell field. Here, we describe a family of synthetically accessible, squaramide-based tripodal supramolecular monomers consisting of a flexible tris(2-aminoethyl)amine (TREN) core that self-assemble into supramolecular polymers and eventually into self-recovering hydrogels. Spectroscopic measurements revealed that monomer aggregation is mainly driven by a combination of hydrogen bonding and hydrophobicity. The self-recovering hydrogels were used to encapsulate NIH 3T3 fibroblasts as well as human-induced pluripotent stem cells (hiPSCs) and their derivatives in 3D. The materials reported here proved cytocompatible for these cell types with maintenance of hiPSCs in their undifferentiated state essential for their subsequent expansion or differentiation into a given cell type and potential for facile release by dilution due to their supramolecular nature. PMID:29528623

Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation.

PubMed

Ouyang, Liliang; Yao, Rui; Mao, Shuangshuang; Chen, Xi; Na, Jie; Sun, Wei

2015-11-04

With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90% ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies.

Measurement of the temperature-dependent threshold shear-stress of red blood cell aggregation.

PubMed

Lim, Hyun-Jung; Nam, Jeong-Hun; Lee, Yong-Jin; Shin, Sehyun

2009-09-01

Red blood cell (RBC) aggregation is becoming an important hemorheological parameter, which typically exhibits temperature dependence. Quite recently, a critical shear-stress was proposed as a new dimensional index to represent the aggregative and disaggregative behaviors of RBCs. The present study investigated the effect of the temperature on the critical shear-stress that is required to keep RBC aggregates dispersed. The critical shear-stress was measured at various temperatures (4, 10, 20, 30, and 37 degrees C) through the use of a transient microfluidic aggregometry. The critical shear-stress significantly increased as the blood temperature lowered, which accorded with the increase in the low-shear blood viscosity with the lowering of the temperature. Furthermore, the critical shear-stress also showed good agreement with the threshold shear-stress, as measured in a rotational Couette flow. These findings assist in rheologically validating the critical shear-stress, as defined in the microfluidic aggregometry.

Optimized suspension culture: the rotating-wall vessel

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Rapid Engineering of Three-Dimensional, Multicellular Tissues With Polymeric Scaffolds

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Jordan, Jacqueline; Fraga, Denise N.

2007-01-01

A process has been developed for the rapid tissue engineering of multicellular-tissue-equivalent assemblies by the controlled enzymatic degradation of polymeric beads in a low-fluid-shear bioreactor. In this process, the porous polymeric beads serve as temporary scaffolds to support the assemblies of cells in a tissuelike 3D configuration during the critical initial growth phases of attachment of anchorage-dependent cells, aggregation of the cells, and formation of a 3D extracellular matrix. Once the cells are assembled into a 3D array and enmeshed in a structural supportive 3D extracellular matrix (ECM), the polymeric scaffolds can be degraded in the low-fluid-shear environment of the NASA-designed bioreactor. The natural 3D tissuelike assembly, devoid of any artificial support structure, is maintained in the low-shear bioreactor environment by the newly formed natural cellular/ECM. The elimination of the artificial scaffold allows normal tissue structure and function.

The bioink: A comprehensive review on bioprintable materials.

PubMed

Hospodiuk, Monika; Dey, Madhuri; Sosnoski, Donna; Ozbolat, Ibrahim T

This paper discusses "bioink", bioprintable materials used in three dimensional (3D) bioprinting processes, where cells and other biologics are deposited in a spatially controlled pattern to fabricate living tissues and organs. It presents the first comprehensive review of existing bioink types including hydrogels, cell aggregates, microcarriers and decellularized matrix components used in extrusion-, droplet- and laser-based bioprinting processes. A detailed comparison of these bioink materials is conducted in terms of supporting bioprinting modalities and bioprintability, cell viability and proliferation, biomimicry, resolution, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and post-bioprinting maturation times, tissue fusion and formation post-implantation, degradation characteristics, commercial availability, immune-compatibility, and application areas. The paper then discusses current limitations of bioink materials and presents the future prospects to the reader. Copyright © 2017 Elsevier Inc. All rights reserved.

Three-dimensional nano-biointerface as a new platform for guiding cell fate.

PubMed

Liu, Xueli; Wang, Shutao

2014-04-21

Three-dimensional nano-biointerface has been emerging as an important topic for chemistry, nanotechnology, and life sciences in recent years. Understanding the exchanges of materials, signals, and energy at biological interfaces has inspired and helped the serial design of three-dimensional nano-biointerfaces. The intimate interactions between cells and nanostructures bring many novel properties, making three-dimensional nano-biointerfaces a powerful platform to guide cell fate in a controllable and accurate way. These advantages and capabilities endow three-dimensional nano-biointerfaces with an indispensable role in developing advanced biological science and technology. This tutorial review is mainly focused on the recent progress of three-dimensional nano-biointerfaces and highlights the new explorations and unique phenomena of three-dimensional nano-biointerfaces for cell-related fundamental studies and biomedical applications. Some basic bio-inspired principles for the design and creation of three-dimensional nano-biointerfaces are also delivered in this review. Current and further challenges of three-dimensional nano-biointerfaces are finally addressed and proposed.

Applications of Fluorogens with Rotor Structures in Solar Cells.

PubMed

Ong, Kok-Haw; Liu, Bin

2017-05-29

Solar cells are devices that convert light energy into electricity. To drive greater adoption of solar cell technologies, higher cell efficiencies and reductions in manufacturing cost are necessary. Fluorogens containing rotor structures may be helpful in addressing some of these challenges due to their unique twisted structures and photophysics. In this review, we discuss the applications of rotor-containing molecules as dyes for luminescent down-shifting layers and luminescent solar concentrators, where their aggregation-induced emission properties and large Stokes shifts are highly desirable. We also discuss the applications of molecules containing rotors in third-generation solar cell technologies, namely dye-sensitized solar cells and organic photovoltaics, where the twisted 3-dimensional rotor structures are used primarily for aggregation control. Finally, we discuss perspectives on the future role of molecules containing rotor structures in solar cell technologies.

Scaffold-free Tissue Formation Under Real and Simulated Microgravity Conditions.

PubMed

Aleshcheva, Ganna; Bauer, Johann; Hemmersbach, Ruth; Slumstrup, Lasse; Wehland, Markus; Infanger, Manfred; Grimm, Daniela

2016-10-01

Scaffold-free tissue formation in microgravity is a new method in regenerative medicine and an important topic in Space Medicine. In this MiniReview, we focus on recent findings in the field of tissue engineering that were observed by exposing cells to real microgravity in space or to devices simulating to at least some extent microgravity conditions on Earth (ground-based facilities). Under both conditions - real and simulated microgravity - a part of the cultured cells of various populations detaches from the bottom of a culture flask. The cells form three-dimensional (3D) aggregates resembling the organs from which the cells have been derived. As spaceflights are rare and extremely expensive, cell culture under simulated microgravity allows more comprehensive and frequent studies on the scaffold-free 3D tissue formation in some aspects, as a number of publications have proven during the last two decades. In this MiniReview, we summarize data from our own studies and work from various researchers about tissue engineering of multi-cellular spheroids formed by cancer cells, tube formation by endothelial cells and cartilage formation by exposing the cells to ground-based facilities such as the 3D Random Positioning Machine (RPM), the 2D Fast-Rotating Clinostat (FRC) or the Rotating Wall Vessel (RWV). Subsequently, we investigated self-organization of 3D aggregates without scaffolds pursuing to enhance the frequency of 3D formation and to enlarge the size of the organ-like aggregates. The density of the monolayer exposed to real or simulated microgravity as well as the composition of the culture media revealed an impact on the results. Genomic and proteomic alterations were induced by simulated microgravity. Under microgravity conditions, adherent cells expressed other genes than cells grown in spheroids. In this MiniReview, the recent improvements in scaffold-free tissue formation are summarized and relationships between phenotypic and molecular appearance are highlighted. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Radar signatures of snowflake riming: A modeling study.

PubMed

Leinonen, Jussi; Szyrmer, Wanda

2015-08-01

The capability to detect the state of snowflake riming reliably from remote measurements would greatly expand the understanding of its global role in cloud-precipitation processes. To investigate the ability of multifrequency radars to detect riming, a three-dimensional model of snowflake growth was used to generate simulated aggregate and crystal snowflakes with various degrees of riming. Three different growth scenarios, representing different temporal relationships between aggregation and riming, were formulated. The discrete dipole approximation was then used to compute the radar backscattering properties of the snowflakes at frequencies of 9.7, 13.6, 35.6, and 94 GHz. In two of the three growth scenarios, the rimed snowflakes exhibit large differences between the backscattering cross sections of the detailed three-dimensional models and the equivalent homogeneous spheroidal models, similarly to earlier results for unrimed snowflakes. When three frequencies are used simultaneously, riming appears to be detectable in a robust manner across all three scenarios. In spite of the differences in backscattering cross sections, the triple-frequency signatures of heavily rimed particles resemble those of the homogeneous spheroids, thus explaining earlier observational results that were compatible with such spheroids.

Magnetic resonance imaging (MRI) and relaxation time mapping of concrete

NASA Astrophysics Data System (ADS)

Beyea, Steven Donald

2001-07-01

The use of Magnetic Resonance Imaging (MRI) of water in concrete is presented. This thesis will approach the problem of MR imaging of concrete by attempting to design new methods, suited to concrete materials, rather than attempting to force the material to suit the method. A number of techniques were developed, which allow the spatial observation of water in concrete in up to three dimensions, and permits the determination of space resolved moisture content, as well as local NMR relaxation times. These methods are all based on the Single-Point Imaging (SPI) method. The development of these new methods will be described, and the techniques validated using phantom studies. The study of one-dimensional moisture transport in drying concrete was performed using SPI. This work examined the effect of initial mixture proportions and hydration time on the drying behaviour of concrete, over a period of three months. Studies of drying concrete were also performed using spatial mapping of the spin-lattice (T1) and effective spin-spin (T2*) relaxation times, thereby permitting the observation of changes in the water occupied pore surface-to-volume ratio (S/V) as a function of drying. Results of this work demonstrated changes in the S/V due to drying, hydration and drying induced microcracking. Three-dimensional MRI of concrete was performed using SPRITE (Single-Point Ramped Imaging with T1 Enhancement) and turboSPI (turbo Single Point Imaging). While SPRITE allows for weighting of MR images using T 1 and T2*, turboSPI allows T2 weighting of the resulting images. Using relaxation weighting it was shown to be possible to discriminate between water contained within a hydrated cement matrix, and water in highly porous aggregates, used to produce low-density concrete. Three dimensional experiments performed using SPRITE and turboSPI examined the role of self-dessication, drying, initial aggregate saturation and initial mixture conditions on the transport of moisture between porous aggregates and the hydrated matrix. The results demonstrate that water is both added and removed from the aggregates, depending upon the physical conditions. The images also appear to show an influx of cement products into cracks in the solid aggregate. (Abstract shortened by UMI.)

Titanium-Enriched Hydroxyapatite–Gelatin Scaffolds with Osteogenically Differentiated Progenitor Cell Aggregates for Calvaria Bone Regeneration

PubMed Central

Padilla, Ricardo; Urkasemsin, Ganokon; Yoon, Kun; Goeckner, Kelly; Hu, Wei-Shou

2013-01-01

Adequate bony support is the key to re-establish both function and esthetics in the craniofacial region. Autologous bone grafting has been the gold standard for regeneration of problematic large bone defects. However, poor graft availability and donor-site complications have led to alternative bone tissue-engineering approaches combining osteoinductive biomaterials and three-dimensional cell aggregates in scaffolds or constructs. The goal of the present study was to generate novel cell aggregate-loaded macroporous scaffolds combining the osteoinductive properties of titanium dioxide (TiO2) with hydroxyapatite–gelatin nanocomposites (HAP-GEL) for regeneration of craniofacial defects. Here we investigated the in vivo applicability of macroporous (TiO2)-enriched HAP-GEL scaffolds with undifferentiated and osteogenically differentiated multipotent adult progenitor cell (MAPC and OD-MAPC, respectively) aggregates for calvaria bone regeneration. The silane-coated HAP-GEL with and without TiO2 additives were polymerized and molded to produce macroporous scaffolds. Aggregates of the rat MAPC were precultured, loaded into each scaffold, and implanted to rat calvaria critical-size defects to study bone regeneration. Bone autografts were used as positive controls and a poly(lactic-co-glycolic acid) (PLGA) scaffold for comparison purposes. Preimplanted scaffolds and calvaria bone from pig were tested for ultimate compressive strength with an Instron 4411® and for porosity with microcomputerized tomography (μCT). Osteointegration and newly formed bone (NFB) were assessed by μCT and nondecalcified histology, and quantified by calcium fluorescence labeling. Results showed that the macroporous TiO2-HAP-GEL scaffold had a comparable strength relative to the natural calvaria bone (13.8±4.5 MPa and 24.5±8.3 MPa, respectively). Porosity was 1.52±0.8 mm and 0.64±0.4 mm for TiO2-HAP-GEL and calvaria bone, respectively. At 8 and 12 weeks postimplantation into rat calvaria defects, greater osteointegration and NFB were significantly present in the TiO2-enriched HAP-GEL constructs with OD-MAPCs, compared to the undifferentiated MAPC-loaded constructs, cell-free HAP-GEL with and without titanium, and PLGA scaffolds. The tissue-engineered TiO2-enriched HAP-GEL constructs with OD-MAPC aggregates present a potential useful therapeutic approach for calvaria bone regeneration. PMID:23495972

Three cyanobacteriochromes work together to form a light color-sensitive input system for c-di-GMP signaling of cell aggregation.

PubMed

Enomoto, Gen; Ni-Ni-Win; Narikawa, Rei; Ikeuchi, Masahiko

2015-06-30

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that have diverse spectral properties and domain compositions. Although large numbers of CBCR genes exist in cyanobacterial genomes, no studies have assessed whether multiple CBCRs work together. We recently showed that the diguanylate cyclase (DGC) activity of the CBCR SesA from Thermosynechococcus elongatus is activated by blue-light irradiation and that, when irradiated, SesA, via its product cyclic dimeric GMP (c-di-GMP), induces aggregation of Thermosynechococcus vulcanus cells at a temperature that is suboptimum for single-cell viability. For this report, we first characterize the photobiochemical properties of two additional CBCRs, SesB and SesC. Blue/teal light-responsive SesB has only c-di-GMP phosphodiesterase (PDE) activity, which is up-regulated by teal light and GTP. Blue/green light-responsive SesC has DGC and PDE activities. Its DGC activity is enhanced by blue light, whereas its PDE activity is enhanced by green light. A ΔsesB mutant cannot suppress cell aggregation under teal-green light. A ΔsesC mutant shows a less sensitive cell-aggregation response to ambient light. ΔsesA/ΔsesB/ΔsesC shows partial cell aggregation, which is accompanied by the loss of color dependency, implying that a nonphotoresponsive DGC(s) producing c-di-GMP can also induce the aggregation. The results suggest that SesB enhances the light color dependency of cell aggregation by degrading c-di-GMP, is particularly effective under teal light, and, therefore, seems to counteract the induction of cell aggregation by SesA. In addition, SesC seems to improve signaling specificity as an auxiliary backup to SesA/SesB activities. The coordinated action of these three CBCRs highlights why so many different CBCRs exist.

Version 4.0 of code Java for 3D simulation of the CCA model

NASA Astrophysics Data System (ADS)

Fan, Linyu; Liao, Jianwei; Zuo, Junsen; Zhang, Kebo; Li, Chao; Xiong, Hailing

2018-07-01

This paper presents a new version Java code for the three-dimensional simulation of Cluster-Cluster Aggregation (CCA) model to replace the previous version. Many redundant traverses of clusters-list in the program were totally avoided, so that the consumed simulation time is significantly reduced. In order to show the aggregation process in a more intuitive way, we have labeled different clusters with varied colors. Besides, a new function is added for outputting the particle's coordinates of aggregates in file to benefit coupling our model with other models.

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.

The roles of carbohydrates, proteins and lipids in the process of aggregation of natural marine organic matter investigated by means of 2D correlation spectroscopy applied to infrared spectra.

PubMed

Mecozzi, Mauro; Pietrantonio, Eva; Pietroletti, Marco

2009-01-01

In this paper the marine organic matter soluble in an alkaline medium called extractable humic substance (EHS), was extracted from three sediment samples of Tyrrhenian Sea and separated by precipitation at pH 2 in the two fractions of fulvic acids (FAs) and humic acids (HAs). FAs were further fractionated in seven sub-samples of different molecular weight (mw) by means of seven different ultrafiltration membranes operating in the range between mw<1 kDa and mw>100 kDa. Then the qualitative composition of each sample of fractionated FAs and HAs was studied by means of one-dimensional Fourier transform infrared spectroscopy in reflectance mode (FTIR-DRIFT) and by two-dimensional (2D) correlation spectroscopy both in wavelength-wavelength (WW) and in sample-sample (SS) mode. The application of 2D correlation WW spectroscopy allows to elucidate the different roles played by carbohydrates and proteins with respect to some lipid compounds such as fatty acids and ester fatty acids during the process of aggregate formations from mw approximately 1 kDa to higher size aggregates. In addition, 2D correlation WW spectroscopy allows to observe some peculiar interactions between carbohydrates and proteins in the formation of EHS aggregates, interactions which vary from a sample to another sample. The results of 2D correlation SS spectroscopy confirm the general evidences obtained by 2D WW spectroscopy and moreover, they also describe the formation of EHS aggregates as a complex process where evolutionary links and connectivity between aggregates of neighbour molecular size ranges are not evident. Two-dimensional correlation spectroscopy applied to FTIR spectroscopy shows to be a powerful tool for the investigation of the mechanisms involved in EHS aggregation because it supports the acquisition of structural information which sometimes can be hardly obtained by one-dimensional FTIR spectroscopy.

Learning the Cell Structures with Three-Dimensional Models: Students' Achievement by Methods, Type of School and Questions' Cognitive Level

ERIC Educational Resources Information Center

Lazarowitz, Reuven; Naim, Raphael

2014-01-01

The cell topic was taught to 9th-grade students in three modes of instruction: (a) students "hands-on," who constructed three-dimensional cell organelles and macromolecules during the learning process; (b) teacher demonstration of the three-dimensional model of the cell structures; and (c) teaching the cell topic with the regular…

Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.

PubMed

Dainiak, Maria B; Savina, Irina N; Musolino, Isabella; Kumar, Ashok; Mattiasson, Bo; Galaev, Igor Yu

2008-01-01

Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

PubMed

Grégoire, C; Marco, S; Thimonier, J; Duplan, L; Laurine, E; Chauvin, J P; Michel, B; Peyrot, V; Verdier, J M

2001-07-02

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer's disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer's disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

Three-Dimensional Orientation of Anisotropic Plasmonic Aggregates at Intracellular Nuclear Indentation Sites by Integrated Light Sheet Super-Resolution Microscopy.

PubMed

Chakkarapani, Suresh Kumar; Sun, Yucheng; Lee, Seungah; Fang, Ning; Kang, Seong Ho

2018-05-22

Three-dimensional (3D) orientations of individual anisotropic plasmonic nanoparticles in aggregates were observed in real time by integrated light sheet super-resolution microscopy ( iLSRM). Asymmetric light scattering of a gold nanorod (AuNR) was used to trigger signals based on the polarizer angle. Controlled photoswitching was achieved by turning the polarizer and obtaining a series of images at different polarization directions. 3D subdiffraction-limited super-resolution images were obtained by superlocalization of scattering signals as a function of the anisotropic optical properties of AuNRs. Varying the polarizer angle allowed resolution of the orientation of individual AuNRs. 3D images of individual nanoparticles were resolved in aggregated regions, resulting in as low as 64 nm axial resolution and 28 nm spatial resolution. The proposed imaging setup and localization approach demonstrates a convenient method for imaging under a noisy environment where the majority of scattering noise comes from cellular components. This integrated 3D iLSRM and localization technique was shown to be reliable and useful in the field of 3D nonfluorescence super-resolution imaging.

Pluripotency of adult stem cells derived from human and rat pancreas

NASA Astrophysics Data System (ADS)

Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

Highly cytocompatible and flexible three-dimensional graphene/polydimethylsiloxane composite for culture and electrochemical detection of L929 fibroblast cells.

PubMed

Waiwijit, Uraiwan; Maturos, Thitima; Pakapongpan, Saithip; Phokharatkul, Ditsayut; Wisitsoraat, Anurat; Tuantranont, Adisorn

2016-08-01

Recently, three-dimensional graphene interconnected network has attracted great interest as a scaffold structure for tissue engineering due to its high biocompatibility, high electrical conductivity, high specific surface area and high porosity. However, free-standing three-dimensional graphene exhibits poor flexibility and stability due to ease of disintegration during processing. In this work, three-dimensional graphene is composited with polydimethylsiloxane to improve the structural flexibility and stability by a new simple two-step process comprising dip coating of polydimethylsiloxane on chemical vapor deposited graphene/Ni foam and wet etching of nickel foam. Structural characterizations confirmed an interconnected three-dimensional multi-layer graphene structure with thin polydimethylsiloxane scaffold. The composite was employed as a substrate for culture of L929 fibroblast cells and its cytocompatibility was evaluated by cell viability (Alamar blue assay), reactive oxygen species production and vinculin immunofluorescence imaging. The result revealed that cell viability on three-dimensional graphene/polydimethylsiloxane composite increased with increasing culture time and was slightly different from a polystyrene substrate (control). Moreover, cells cultured on three-dimensional graphene/polydimethylsiloxane composite generated less ROS than the control at culture times of 3-6 h. The results of immunofluorescence staining demonstrated that fibroblast cells expressed adhesion protein (vinculin) and adhered well on three-dimensional graphene/polydimethylsiloxane surface. Good cell adhesion could be attributed to suitable surface properties of three-dimensional graphene/polydimethylsiloxane with moderate contact angle and small negative zeta potential in culture solution. The results of electrochemical study by cyclic voltammetry showed that an oxidation current signal with no apparent peak was induced by fibroblast cells and the oxidation current at an oxidation potential of +0.9 V increased linearly with increasing cell number. Therefore, the three-dimensional graphene/polydimethylsiloxane composite exhibits high cytocompatibility and can potentially be used as a conductive substrate for cell-based electrochemical sensing. © The Author(s) 2016.

Spontaneous Contractility-Mediated Cortical Flow Generates Cell Migration in Three-Dimensional Environments

PubMed Central

Hawkins, Rhoda J.; Poincloux, Renaud; Bénichou, Olivier; Piel, Matthieu; Chavrier, Philippe; Voituriez, Raphaël

2011-01-01

We present a model of cell motility generated by actomyosin contraction of the cell cortex. We identify, analytically, dynamical instabilities of the cortex and show that they yield steady-state cortical flows, which, in turn, can induce cell migration in three-dimensional environments. This mechanism relies on the regulation of contractility by myosin, whose transport is explicitly taken into account in the model. Theoretical predictions are compared to experimental data of tumor cells migrating in three-dimensional matrigel and suggest that this mechanism could be a general mode of cell migration in three-dimensional environments. PMID:21889440

Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration.

PubMed

Kim, Min-Cheol; Silberberg, Yaron R; Abeyaratne, Rohan; Kamm, Roger D; Asada, H Harry

2018-01-16

Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.

Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression.

PubMed

Dolega, M E; Delarue, M; Ingremeau, F; Prost, J; Delon, A; Cappello, G

2017-01-27

The surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core.

Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation.

PubMed

Sart, Sébastien; Ma, Teng; Li, Yan

2013-01-01

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate-based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size-dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein-free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three-germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein-free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Induction of Three-Dimensional Growth of Human Liver Cells in Simulated Microgravity

NASA Technical Reports Server (NTRS)

Pellis, Neal R.; Khaoustov, V. I.; Yoffe, B.; Murry, D. J.; Soriano, H. E.; Risin, D.; Dawson, David L. (Technical Monitor)

1999-01-01

We previously reported that a NASA-developed bioreactor that simulates microgravity environment and creates the unique environment of low shear force and high-mass transfer is conducive for maintaining long term 3-D cell cultures of functional hepatocytes (60 days). However, significant further expansion of liver mass, or the remodeling of liver in vitro was jeopardized by the appearance of apoptotic zones in the center of large cell aggregates. To optimize oxygenation and nutritional uptake within growing cellular aggregates we cultured primary human liver cells (HLC) in a bioreactor in the presence or absence of microcarriers and biodegradable scaffolds. Also, to promote angiogenesis, HLC were cultured with or without microvascular endothelial cells. HLC were harvested from human livers by collagenase perfusion. While microcarriers did not affect cell growth, HLC cultured with biodegradable scaffolds made from polyglycolic acid (PGA) formed aggregates up to 3 cm in length. Culturing cells with PGA scaffolds increased the efficiency of cell self-assembly and the formation of larger cell aggregates. Based on histological evaluation it appears that the degree of apoptotic cells was diminished as compared to cultures without scaffolds. Histology of HLC with PGA-scaffolds revealed cell distribution between the fibers of the scaffolds, and cell-cell and cell-fiber interactions. Analyses of HLC spheroids revealed tissue-like structures comprised of hepatocytes, biliary epithelial cells and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes and bile canaliculi with multiple microvilli and tight cellular junctions. Hepatocytes were further organized into tight clusters surrounded by complex stromal structures and reticulin fibers. Also, we observed higher levels of albumin mRNA expression when hepatocytes were co-cultured with endothelial cells. To evaluate viability and microsomal deethylation activity of hepatocytes we assessed the metabolism of midazolam by gas chromatography. Samples that were collected from HLC cultured in the bioreactor contained higher levels of midazolam metabolites (1-OH-midazolam and 4-OH-midazolam) than samples collected from conventional cultures. In summary, we have shown that co-culture of HLC with endothelial cells and/or culturing in the presence of PGA scaffolds provides additional advantages in maintaining functional activity of 3-D hepatocyte cultures that resemble liver tissue. This cell culture system may facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo and suggest the feasibility of using this approach for pre-clinical metabolic screening of novel drug candidates.

Three Dimensional Primary Hepatocyte Culture

NASA Technical Reports Server (NTRS)

Yoffe, Boris

1998-01-01

Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate.

PubMed

Altmann, Brigitte; Steinberg, Thorsten; Giselbrecht, Stefan; Gottwald, Eric; Tomakidi, Pascal; Bächle-Haas, Maria; Kohal, Ralf-Joachim

2011-12-01

Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.

Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells

PubMed Central

Turner, David A.; Hayward, Penelope C.; Baillie-Johnson, Peter; Rué, Pau; Broome, Rebecca; Faunes, Fernando; Martinez Arias, Alfonso

2014-01-01

The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/β-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation. PMID:25371361

Creating physically-based three-dimensional microstructures: Bridging phase-field and crystal plasticity models.

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

Lim, Hojun; Owen, Steven J.; Abdeljawad, Fadi F.

In order to better incorporate microstructures in continuum scale models, we use a novel finite element (FE) meshing technique to generate three-dimensional polycrystalline aggregates from a phase field grain growth model of grain microstructures. The proposed meshing technique creates hexahedral FE meshes that capture smooth interfaces between adjacent grains. Three dimensional realizations of grain microstructures from the phase field model are used in crystal plasticity-finite element (CP-FE) simulations of polycrystalline a -iron. We show that the interface conformal meshes significantly reduce artificial stress localizations in voxelated meshes that exhibit the so-called "wedding cake" interfaces. This framework provides a direct linkmore » between two mesoscale models - phase field and crystal plasticity - and for the first time allows mechanics simulations of polycrystalline materials using three-dimensional hexahedral finite element meshes with realistic topological features.« less

Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

2012-03-01

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

Revealing sub-μm and μm-scale textures in H2O ice at megabar pressures by time-domain Brillouin scattering

PubMed Central

Nikitin, Sergey M.; Chigarev, Nikolay; Tournat, Vincent; Bulou, Alain; Gasteau, Damien; Castagnede, Bernard; Zerr, Andreas; Gusev, Vitalyi E.

2015-01-01

The time-domain Brillouin scattering technique, also known as picosecond ultrasonic interferometry, allows monitoring of the propagation of coherent acoustic pulses, having lengths ranging from nanometres to fractions of a micrometre, in samples with dimension of less than a micrometre to tens of micrometres. In this study, we applied this technique to depth-profiling of a polycrystalline aggregate of ice compressed in a diamond anvil cell to megabar pressures. The method allowed examination of the characteristic dimensions of ice texturing in the direction normal to the diamond anvil surfaces with sub-micrometre spatial resolution via time-resolved measurements of the propagation velocity of the acoustic pulses travelling in the compressed sample. The achieved imaging of ice in depth and in one of the lateral directions indicates the feasibility of three-dimensional imaging and quantitative characterisation of the acoustical, optical and acousto-optical properties of transparent polycrystalline aggregates in a diamond anvil cell with tens of nanometres in-depth resolution and a lateral spatial resolution controlled by pump laser pulses focusing, which could approach hundreds of nanometres. PMID:25790808

Principles for problem aggregation and assignment in medium scale multiprocessors

NASA Technical Reports Server (NTRS)

Nicol, David M.; Saltz, Joel H.

1987-01-01

One of the most important issues in parallel processing is the mapping of workload to processors. This paper considers a large class of problems having a high degree of potential fine grained parallelism, and execution requirements that are either not predictable, or are too costly to predict. The main issues in mapping such a problem onto medium scale multiprocessors are those of aggregation and assignment. We study a method of parameterized aggregation that makes few assumptions about the workload. The mapping of aggregate units of work onto processors is uniform, and exploits locality of workload intensity to balance the unknown workload. In general, a finer aggregate granularity leads to a better balance at the price of increased communication/synchronization costs; the aggregation parameters can be adjusted to find a reasonable granularity. The effectiveness of this scheme is demonstrated on three model problems: an adaptive one-dimensional fluid dynamics problem with message passing, a sparse triangular linear system solver on both a shared memory and a message-passing machine, and a two-dimensional time-driven battlefield simulation employing message passing. Using the model problems, the tradeoffs are studied between balanced workload and the communication/synchronization costs. Finally, an analytical model is used to explain why the method balances workload and minimizes the variance in system behavior.

Measurement of net electric charge and dipole moment of dust aggregates in a complex plasma.

PubMed

Yousefi, Razieh; Davis, Allen B; Carmona-Reyes, Jorge; Matthews, Lorin S; Hyde, Truell W

2014-09-01

Understanding the agglomeration of dust particles in complex plasmas requires knowledge of basic properties such as the net electrostatic charge and dipole moment of the dust. In this study, dust aggregates are formed from gold-coated mono-disperse spherical melamine-formaldehyde monomers in a radiofrequency (rf) argon discharge plasma. The behavior of observed dust aggregates is analyzed both by studying the particle trajectories and by employing computer models examining three-dimensional structures of aggregates and their interactions and rotations as induced by torques arising from their dipole moments. These allow the basic characteristics of the dust aggregates, such as the electrostatic charge and dipole moment, as well as the external electric field, to be determined. It is shown that the experimental results support the predicted values from computer models for aggregates in these environments.

Unsteady Flow Interactions Between Pitching Wings In Schooling Arrangements

NASA Astrophysics Data System (ADS)

Kurt, Melike; Moored, Keith

2017-11-01

In nature, many fish aggregate into large groups or schools for protection against predators, for social interactions and to save energy during migrations. Regardless of their prime motivation, fish experience three-dimensional flow interactions amongst themselves that can improve or hamper swimming performance and give rise to fluid-mediated forces between individuals. To date, the unsteady, three-dimensional flow interactions among schooling fish remains relatively unexplored. In order to study these interactions, the caudal fins of two interacting fish are idealized as two finite span pitching wings arranged in mixtures of canonical in-line and side-by-side arrangements. The forces and moments acting on the wings in the streamwise and cross-stream directions are quantified as the arrangement and the phase delay between the wings is altered. Particle image velocimetry is employed to characterize the flow physics during high efficiency locomotion. Finally, the forces and flowfields of two-dimensional pitching wings are compared with three-dimensional wings to distinguish how three-dimensionality alters the flow interactions in schools of fish.

Crystallography and Molecular Arrangement of Polymorphic Monolayer J-Aggregates of a Cyanine Dye: Multiangle Polarized Light Fluorescence Optical Microscopy Study.

PubMed

Prokhorov, Valery V; Pozin, Sergey I; Perelygina, Olga M; Mal'tsev, Eugene I

2018-04-24

The molecular orientation in monolayer J-aggregates of 3,3-di(γ-sulfopropyl)-5,5-dichlorotiamonomethinecyanine dye has been precisely estimated using improved linear polarization measurements in the fluorescence microscope in which a multiangle set of polarization data is obtained using sample rotation. The estimated molecular orientation supplemented with the previously established crystallographic constraints based on the analysis of the well-developed two-dimensional J-aggregate shapes unambiguously indicate the staircase type of molecular arrangement for striplike J-aggregates with the staircases oriented along strips. The molecular transition dipoles are inclined at an angle of ∼25° to the strip direction, whereas the characteristic strip vertex angle ∼45° is formed by the [100] and [1-10] directions of the monoclinic unit cell. Measurements of the geometry of partially unwound tubes and their polarization properties support the model of tube formation by close-packed helical winding of flexible monolayer strips. In the tubes, the long molecular axes are oriented at a small angle in the range of 5-15° to the normal to the tube axis providing low bending energy. At a nanoscale, high-resolution atomic force microscopy imaging of J-aggregate monolayers reveals a complex quasi-one-dimensional organization.

In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

PubMed

Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

2016-03-01

Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.

Three-dimensional organization of dermal fibroblasts by macromass culture.

PubMed

Deshpande, Manisha

2008-01-01

The three-dimensional organization of cells by high-cell-seeding-density culture, termed 'macromass culture', is described. By macromass culture, dermal fibroblasts can be made to organize themselves into a unified three-dimensional form without the aid of a scaffold, and macroscopic constructs, named macromasses, can be made wholly from cells. The sole factor causing three-dimensional organization is culture of cells at high cell seeding density per unit area. No scaffold or extraneous matrix is used for the generation of macromasses; they are of completely cellular origin. No other agents or external influences such as tissue-inducing chemicals, tissue-inducing growth factors, substratum with special properties, rotational culture, centrifugation etc. are employed for macromass formation, and all seeded cells become part of the cohesive construct. These three-dimensional constructs have the potential for use as in vitro tissue analogues, and a possible application for in vitro cytotoxicity testing is demonstrated.

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

PubMed

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

1999-02-01

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

Mimicking Metastases Including Tumor Stroma: A New Technique to Generate a Three-Dimensional Colorectal Cancer Model Based on a Biological Decellularized Intestinal Scaffold.

PubMed

Nietzer, Sarah; Baur, Florentin; Sieber, Stefan; Hansmann, Jan; Schwarz, Thomas; Stoffer, Carolin; Häfner, Heide; Gasser, Martin; Waaga-Gasser, Ana Maria; Walles, Heike; Dandekar, Gudrun

2016-07-01

Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of β-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions.

Computational Selection of Inhibitors of A-beta Aggregation and Neuronal Toxicity

PubMed Central

Chen, Deliang; Martin, Zane S.; Soto, Claudio; Schein, Catherine H.

2009-01-01

Alzheimer’s Disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-β protein (Aβ). Disease symptoms can be alleviated, in vitro and in vivo, by “β-sheet breaker” pentapeptides that reduce plaque volume. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related β-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Aβ. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features in a conformation similar to the active peptides were selected, ranked by docking solubility parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Aβ aggregation at 2–3 μM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Aβ on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD. PMID:19540126

Etude expérimentale de la lamination des stromatolithes à Rivularia haematites en climat tempéré: édification des lamines micritiques

NASA Astrophysics Data System (ADS)

Caudwell, Christiane; Lang, Jacques; Pascal, André

1997-06-01

The lamination of Rivularia haematites stromatolites (D.C.) Agardh was studied experimentally for 7 years. Micritic laminae are found to form in three stages: biological formation of dark laminae during the wet season, microsparitic calcification of these laminae in the form of clearly individualized polycrystalline aggregates and, finally, micritization of the latter by bacterial action. These three stages develop over 2 to 3 years. The occurrence of transverse, longitudinal and circular microfibrils in the outer sheath is thought to explain the nucleation and the three-dimensional structure of the microsparitic crystals of the dark laminae and of the polycrystalline aggregates.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer’s disease

PubMed Central

Grégoire, Catherine; Marco, Sergio; Thimonier, Jean; Duplan, Laure; Laurine, Emmanuelle; Chauvin, Jean-Paul; Michel, Bernard; Peyrot, Vincent; Verdier, Jean-Michel

2001-01-01

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer’s disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer’s disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils. PMID:11432819

High resolution microscopy reveals the nuclear shape of budding yeast during cell cycle and in various biological states

PubMed Central

Kamgoue, Alain; Normand, Christophe; Léger-Silvestre, Isabelle; Mangeat, Thomas

2016-01-01

ABSTRACT How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope. PMID:27831493

Three-dimensional confocal microscopy of the living cornea and ocular lens

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-07-01

The three-dimensional reconstruction of the optic zone of the cornea and the ocular crystalline lens has been accomplished using confocal microscopy and volume rendering computer techniques. A laser scanning confocal microscope was used in the reflected light mode to obtain the two-dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133 three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their 'beaded' cell borders, basal lamina, nerve plexus, nerve fibers, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in- situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers. The three-dimensional data sets of the cornea and the ocular lens were reconstructed in the computer using volume rendering techniques. Stereo pairs were also created of the two- dimensional ocular images for visualization. The stack of two-dimensional images was reconstructed into a three-dimensional object using volume rendering techniques. This demonstration of the three-dimensional visualization of the intact, enucleated eye provides an important step toward quantitative three-dimensional morphometry of the eye. The important aspects of three-dimensional reconstruction are discussed.

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

PubMed

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

2018-02-01

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

Long-Term Results of Cartilage Repair after Allogeneic Transplantation of Cartilaginous Aggregates Formed from Bone Marrow-Derived Cells for Large Osteochondral Defects in Rabbit Knees.

PubMed

Yoshioka, Tomokazu; Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa

2013-10-01

The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells. Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow-derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O'Driscoll score). The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O'Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Red blood cell deformability and aggregation behaviour in different animal species.

PubMed

Plasenzotti, R; Stoiber, B; Posch, M; Windberger, U

2004-01-01

Comparative animal studies showed the wide variation of whole blood and plasma viscosity, and erythrocyte aggregation among mammalian species. Whole blood viscosity and red blood cell aggregation is influenced by red cell fluidity. To evaluate differences in erythrocyte deformability in mammals, three species were investigated, whose erythrocytes have a different aggregation property: horse, as a species with high, dog with medium, and sheep with almost unmeasurable aggregation tendency. Erythrocyte deformability was tested ektacytometrically (Elongation Index [EI], LORCA, Mechatronics, Hoorn, Netherlands) at shear stresses from 0.30 to 53.06 Pa. Equine erythrocytes showed EI-values from 0.047 at low shear stress to 0.541 at high shear stress. The EI from dog's erythrocytes ranged from 0.035 to 0.595. Sheep's erythrocytes had an EI of 0.005 at low and 0.400 at high shear stress. Although it might be presumed from the aggregation property that horse had the highest EI among the three species, the EI of canine erythrocytes exceeded the value in horses by 10% at high shear stress. Further, equine erythrocytes started to deform at higher shear stresses (1.69 Pa) than did canine and ovine cells, whose EI increased continuously with increasing shear stress. At moderate shear stress (1-5 Pa) deformability was even higher in the sheep than in the horse. However, at shear stresses higher than 5.34 Pa, equine red cell elongation clearly exceeded the values of sheep. We conclude that erythrocyte elongation is different between the animal species, not clearly linked with the aggregation property, and that the degree of deformability at various shear stresses is species-specific.

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells

PubMed Central

Rodriguez, Jose A.; Xu, Rui; Chen, Chien-Chun; Huang, Zhifeng; Jiang, Huaidong; Chen, Allan L.; Raines, Kevin S.; Pryor Jr, Alan; Nam, Daewoong; Wiegart, Lutz; Song, Changyong; Madsen, Anders; Chushkin, Yuriy; Zontone, Federico; Bradley, Peter J.; Miao, Jianwei

2015-01-01

A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 keV X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and the three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. It is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres. PMID:26306199

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells

DOE PAGES

Rodriguez, Jose A.; Xu, Rui; Chen, Chien -Chun; ...

2015-09-01

Here, a structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 Kev X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and themore » three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. Finally, it is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres.« less

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells.

PubMed

Rodriguez, Jose A; Xu, Rui; Chen, Chien-Chun; Huang, Zhifeng; Jiang, Huaidong; Chen, Allan L; Raines, Kevin S; Pryor, Alan; Nam, Daewoong; Wiegart, Lutz; Song, Changyong; Madsen, Anders; Chushkin, Yuriy; Zontone, Federico; Bradley, Peter J; Miao, Jianwei

2015-09-01

A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 keV X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and the three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. It is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres.

Three-dimensional multi-scale model of deformable platelets adhesion to vessel wall in blood flow

PubMed Central

Wu, Ziheng; Xu, Zhiliang; Kim, Oleg; Alber, Mark

2014-01-01

When a blood vessel ruptures or gets inflamed, the human body responds by rapidly forming a clot to restrict the loss of blood. Platelets aggregation at the injury site of the blood vessel occurring via platelet–platelet adhesion, tethering and rolling on the injured endothelium is a critical initial step in blood clot formation. A novel three-dimensional multi-scale model is introduced and used in this paper to simulate receptor-mediated adhesion of deformable platelets at the site of vascular injury under different shear rates of blood flow. The novelty of the model is based on a new approach of coupling submodels at three biological scales crucial for the early clot formation: novel hybrid cell membrane submodel to represent physiological elastic properties of a platelet, stochastic receptor–ligand binding submodel to describe cell adhesion kinetics and lattice Boltzmann submodel for simulating blood flow. The model implementation on the GPU cluster significantly improved simulation performance. Predictive model simulations revealed that platelet deformation, interactions between platelets in the vicinity of the vessel wall as well as the number of functional GPIbα platelet receptors played significant roles in platelet adhesion to the injury site. Variation of the number of functional GPIbα platelet receptors as well as changes of platelet stiffness can represent effects of specific drugs reducing or enhancing platelet activity. Therefore, predictive simulations can improve the search for new drug targets and help to make treatment of thrombosis patient-specific. PMID:24982253

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2013-03-01

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression.

PubMed

Lewis, Phillip L; Green, Richard M; Shah, Ramille N

2018-03-15

Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in 2D models. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

From two-dimensional graphene oxide to three-dimensional honeycomb-like Ni3S2@graphene oxide composite: insight into structure and electrocatalytic properties

NASA Astrophysics Data System (ADS)

Wei, Xinting; Li, Yueqiang; Xu, Wenli; Zhang, Kaixuan; Yin, Jie; Shi, Shaozhen; Wei, Jiazhen; Di, Fangfang; Guo, Junxue; Wang, Can; Chu, Chaofan; Sui, Ning; Chen, Baoli; Zhang, Yingtian; Hao, Hongguo; Zhang, Xianxi; Zhao, Jinsheng; Zhou, Huawei; Wang, Shuhao

2017-12-01

Three-dimensional (3D) graphene composites have drawn increasing attention in energy storage/conversion applications due to their unique structures and properties. Herein, we synthesized 3D honeycomb-like Ni3S2@graphene oxide composite (3D honeycomb-like Ni3S2@GO) by a one-pot hydrothermal method. We found that positive charges of Ni2+ and negative charges of NO_{3^{- in Ni(NO3)2 induced a transformation of graphene oxide with smooth surface into graphene oxide with wrinkled surface (w-GO). The w-GO in the mixing solution of Ni(NO3)2/thioacetamide/H2O evolved into 3D honeycomb-like Ni3S2@GO in solvothermal process. The GO effectively inhibited the aggregation of Ni3S2 nanoparticles. Photoelectrochemical cells based on 3D Ni3S2@GO synthesized at 60 mM l-1 Ni(NO3)2 exhibited the best energy conversion efficiency. 3D Ni3S2@GO had smaller charge transfer resistance and larger exchange current density than pure Ni3S2 for iodine reduction reaction. The cyclic stability of 3D honeycomb-like Ni3S2@GO was good in the iodine electrolyte. Results are of great interest for fundamental research and practical applications of 3D GO and its composites in solar water-splitting, artificial photoelectrochemical cells, electrocatalysts and Li-S or Na-S batteries.}}

Ultrastructural evaluation of mesenchymal stem cells from inflamed periodontium in different in vitro conditions.

PubMed

Zaganescu, Raluca; Barbu Tudoran, Lucian; Pall, Emoke; Florea, Adrian; Roman, Alexandra; Soanca, Andrada; Mihaela Mihu, Carmen

2015-09-01

This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three-dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro- and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast-like or star-like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells. © 2015 Wiley Periodicals, Inc.

Self-Assembly of Supramolecular Aggregates Based on Sector- and Cone-Shaped Dendrons and Bolaamphiphiles

NASA Astrophysics Data System (ADS)

Shcherbina, M. A.; Chvalun, S. N.

2018-06-01

Using a number of classes of such sector-shaped macromolecules as derivatives of 2,3,4- and 3,4,5- tri(dodecyloxy)benzenesulfonic acid and dendrimers based on gallic acid as an example, the main stages in the formation of supramolecular ensembles are considered: the formation of individual supramolecular aggregates due to the weak noncovalent interactions of mesogenic groups, and the subsequent ordering within these aggregates, which lowers the free energy of a system. Supramolecular aggregates are in turn organized into two- or three-dimensional supramolecular lattices. It is shown that the shape of the supramolecular aggregates and its change along with temperature are functions of the chemical structure of the mesogenic group (resulting in the controlled design of complex self-organizing systems with a given response to external stimuli).

Asp-Gly based peptides confined at the surface of cationic gemini surfactant aggregates.

PubMed

Brizard, Aurélie; Dolain, Christel; Huc, Ivan; Oda, Reiko

2006-04-11

Cationic gemini surfactants complexed with anionic oligoglycine-aspartate (called gemini peptides hereafter) were synthesized, and their aggregation behaviors were studied. The effects of the hydrophobic chain length (C10-C22) and the length of the oligoglycine (0-4) were investigated, and it was clearly shown by critical micellar concentration, Krafft temperature, and isothermal surface pressure measurements that the hydrophobic effect and interpeptidic interaction influence the aggregation behavior in a cooperative manner. Below their Krafft temperatures, some of them formed both hydro- and organogels with three-dimensional networks and the Fourier transform infrared measurements show the presence of interpeptidic hydrogen bonds.

Influence of plankton community structure on the sinking velocity of marine aggregates

NASA Astrophysics Data System (ADS)

Bach, L. T.; Boxhammer, T.; Larsen, A.; Hildebrandt, N.; Schulz, K. G.; Riebesell, U.

2016-08-01

About 50 Gt of carbon is fixed photosynthetically by surface ocean phytoplankton communities every year. Part of this organic matter is reprocessed within the plankton community to form aggregates which eventually sink and export carbon into the deep ocean. The fraction of organic matter leaving the surface ocean is partly dependent on aggregate sinking velocity which accelerates with increasing aggregate size and density, where the latter is controlled by ballast load and aggregate porosity. In May 2011, we moored nine 25 m deep mesocosms in a Norwegian fjord to assess on a daily basis how plankton community structure affects material properties and sinking velocities of aggregates (Ø 80-400 µm) collected in the mesocosms' sediment traps. We noted that sinking velocity was not necessarily accelerated by opal ballast during diatom blooms, which could be due to relatively high porosity of these rather fresh aggregates. Furthermore, estimated aggregate porosity (Pestimated) decreased as the picoautotroph (0.2-2 µm) fraction of the phytoplankton biomass increased. Thus, picoautotroph-dominated communities may be indicative for food webs promoting a high degree of aggregate repackaging with potential for accelerated sinking. Blooms of the coccolithophore Emiliania huxleyi revealed that cell concentrations of 1500 cells/mL accelerate sinking by about 35-40%, which we estimate (by one-dimensional modeling) to elevate organic matter transfer efficiency through the mesopelagic from 14 to 24%. Our results indicate that sinking velocities are influenced by the complex interplay between the availability of ballast minerals and aggregate packaging; both of which are controlled by plankton community structure.

Structural formation of huntingtin-like aggregates probed by small-angle neutron scattering

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

Stanley, Christopher B; Perevozchikova, Tatiana; Berthelier-Jung, Valerie M

2011-01-01

In several neurodegenerative disorders, including Huntington s disease (HD), aspects concerning the earliest of protein structures that form along the aggregation pathway have increasingly gained attention since these particular species are likely to be neurotoxic. We used time-resolved small-angle neutron scattering (SANS) to probe in solution these transient structures formed by peptides having the N-terminal sequence context of mutant huntingtin (Htt) exon 1. We obtained snapshots of the formed aggregates as the kinetic reaction ensued to yield quantitative information on their size and mass. At the early stage, small precursor species with an initial radius of gyration (Rg) of 16.1more » 5.9 and average mass of a dimer to trimer were monitored. Structural growth was treated as two modes with a transition from three-dimensional early aggregate formation to two-dimensional fibril growth and association. Our SANS results on the internal structure of the mature fibrils demonstrate loose packing with about 1 peptide per 4.75 -sheet repeat distance, which is shown to be quantitatively consistent with a -helix model. This research provides new insights into the structures forming along the pathway of Htt exon 1 aggregation and should assist in determining the role that precursors play in neuronal toxicity.« less

The effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients.

PubMed

Bakirdogen, Serkan; Eren, Necmi; Bek, Sibel Gokcay; Mehtap, Ozgur; Cekmen, Mustafa Baki

2016-01-01

Serum leptin levels of chronic kidney disease patients have been detected higher than normal population. The aim of this study was to investigate the effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients. Fourty three peritoneal dialysis patients were included in the study. Thrombocyte aggregation was calculated from the whole blood subsequently the effects of different concentrations of human recombinant leptin on thrombocyte aggregations were investigated. Four test cells were used for this process. While leptin was not added into the first test cell, increasing amounts of leptin was added into the second, third and fourth test cells to attain the concentrations of 25, 50 and 100 ng/ml respectively. Thrombocyte aggregation was inhibited by recombinant leptin in peritoneal dialysis patients. Thrombocyte aggregation mean values were found statistically significantly higher in first test cell when compared to leptin groups in peritoneal dialysis patients. For leptin groups we could not find any statistically significant differences for thrombocyte aggregation mean values between any of the groups. Further studies with larger number of peritoneal dialysis patients are required to prove the action of leptin on thrombocyte aggregation.

The effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients

PubMed Central

Bakirdogen, Serkan; Eren, Necmi; Bek, Sibel Gokcay; Mehtap, Ozgur; Cekmen, Mustafa Baki

2016-01-01

Objective: Serum leptin levels of chronic kidney disease patients have been detected higher than normal population. The aim of this study was to investigate the effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients. Methods: Fourty three peritoneal dialysis patients were included in the study. Thrombocyte aggregation was calculated from the whole blood subsequently the effects of different concentrations of human recombinant leptin on thrombocyte aggregations were investigated. Four test cells were used for this process. While leptin was not added into the first test cell, increasing amounts of leptin was added into the second, third and fourth test cells to attain the concentrations of 25, 50 and 100 ng/ml respectively. Results: Thrombocyte aggregation was inhibited by recombinant leptin in peritoneal dialysis patients. Thrombocyte aggregation mean values were found statistically significantly higher in first test cell when compared to leptin groups in peritoneal dialysis patients. For leptin groups we could not find any statistically significant differences for thrombocyte aggregation mean values between any of the groups. Conclusion: Further studies with larger number of peritoneal dialysis patients are required to prove the action of leptin on thrombocyte aggregation. PMID:28083046

Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors.

PubMed

Almutawaa, Walaa; Rohani, Leili; Rancourt, Derrick E

2016-01-01

Human induced pluripotent stem cells (hiPSCs) hold great promise as a cell source for therapeutic applications and regenerative medicine. Traditionally, hiPSCs are expanded in two-dimensional static culture as colonies in the presence or absence of feeder cells. However, this expansion procedure is associated with lack of reproducibility and low cell yields. To fulfill the large cell number demand for clinical use, robust large-scale production of these cells under defined conditions is needed. Herein, we describe a scalable, low-cost protocol for expanding hiPSCs as aggregates in a lab-scale bioreactor.

Quantitative characterization of 3D deformations of cell interactions with soft biomaterials

NASA Astrophysics Data System (ADS)

Franck, Christian

In recent years, the importance of mechanical forces in directing cellular function has been recognized as a significant factor in biological and physiological processes. In fact, these physical forces are now viewed equally as important as biochemical stimuli in controlling cellular response. Not only do these cellular forces, or cell tractions, play an important role in cell migration, they are also significant to many other physiological and pathological processes, both at the tissue and organ level, including wound healing, inflammation, angiogenesis, and embryogenesis. A complete quantification of cell tractions during cell-material interactions can lead to a deeper understanding of the fundamental role these forces play in cell biology. Thus, understanding the function and role of a cell from a mechanical framework can have important implications towards the development of new implant materials and drug treatments. Previous research has contributed significant descriptions of cell-tissue interactions by quantifying cell tractions in two-dimensional environments; however, most physiological processes are three-dimensional in nature. Recent studies have shown morphological differences in cells cultured on two-dimensional substrates versus three-dimensional matrices, and that the intrinsic extracellular matrix interactions and migration behavior are different in three dimensions versus two dimensions. Hence, measurement techniques are needed to investigate cellular behavior in all three dimensions. This thesis presents a full-field imaging technique capable of quantitatively measuring cell traction forces in all three spatial dimensions, and hence addresses the need of a three-dimensional quantitative imaging technique to gain insight into the fundamental role of physical forces in biological processes. The technique combines laser scanning confocal microscopy (LSCM) with digital volume correlation (DVC) to track the motion of fluorescent particles during cell-induced or externally applied deformations. This method is validated by comparing experimentally measured non-uniform deformation fields near hard and soft spherical inclusions under uniaxial compression with the corresponding analytical solution. Utilization of a newly developed computationally efficient stretch-correlation and deconvolution algorithm is shown to improve the overall measurement accuracy, in particular under large deformations. Using this technique, the full three-dimensional substrate displacement fields are experimentally determined during the migration of individual fibroblast cells on polyacrylamide gels. This is the first study to show the highly three-dimensional structure of cell-induced displacement and traction fields. These new findings suggest a three-dimensional push-pull cell motility, which differs from the traditional theories based on two-dimensional data. These results provide new insight into the dynamic cell-matrix force exchange or mechanotransduction of migrating cells, and will aid in the development of new three-dimensional cell motility and adhesion models. As this study reveals, the mechanical interactions of cells and their extracellular matrix appear to be highly three-dimensional. It also shows that the LSCM-DVC technique is well suited for investigating the mechanics of cell-matrix interactions while providing a platform to access detailed information of the intricate biomechanical coupling for many cellular responses. Thus, this method has the capability to provide direct quantitative experimental data showing how cells interact with their surroundings in three dimensions and might stimulate new avenues of scientific thought in understanding the fundamental role physical forces play in regulating cell behavior.

Formation and structure of stable aggregates in binary diffusion-limited cluster-cluster aggregation processes

NASA Astrophysics Data System (ADS)

López-López, J. M.; Moncho-Jordá, A.; Schmitt, A.; Hidalgo-Álvarez, R.

2005-09-01

Binary diffusion-limited cluster-cluster aggregation processes are studied as a function of the relative concentration of the two species. Both, short and long time behaviors are investigated by means of three-dimensional off-lattice Brownian Dynamics simulations. At short aggregation times, the validity of the Hogg-Healy-Fuerstenau approximation is shown. At long times, a single large cluster containing all initial particles is found to be formed when the relative concentration of the minority particles lies above a critical value. Below that value, stable aggregates remain in the system. These stable aggregates are composed by a few minority particles that are highly covered by majority ones. Our off-lattice simulations reveal a value of approximately 0.15 for the critical relative concentration. A qualitative explanation scheme for the formation and growth of the stable aggregates is developed. The simulations also explain the phenomenon of monomer discrimination that was observed recently in single cluster light scattering experiments.

Cultured High-Fidelity Three-Dimensional Human Urogenital Tract Carcinomas and Process

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Spaulding, Glenn F. (Inventor); Wolf, David A. (Inventor)

1998-01-01

Artificial high-fidelity three-dimensional human urogenital tract carcinomas are propagated under in vitro-microgravity conditions from carcinoma cells. Artificial high-fidelity three-dimensional human urogenital tract carcinomas are also propagated from a coculture of normal urogenital tract cells inoculated with carcinoma cells. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Three-dimensional wet-electrospun poly(lactic acid)/multi-wall carbon nanotubes scaffold induces differentiation of human menstrual blood-derived stem cells into germ-like cells.

PubMed

Eyni, Hossein; Ghorbani, Sadegh; Shirazi, Reza; Salari Asl, Leila; P Beiranvand, Shahram; Soleimani, Masoud

2017-09-01

Infertility caused by the disruption or absence of germ cells is a major and largely incurable medical problem. Germ cells (i.e., sperm or egg) play a key role in the transmission of genetic and epigenetic information across generations. Generation of gametes derived in vitro from stem cells hold promising prospects which could potentially help infertile men and women. Menstrual blood-derived stem cells are a unique stem cell source. Evidence suggests that menstrual blood-derived stem cells exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine. To maintain the three-dimensional structure of natural extra cellular matrices in vitro, scaffolds can do this favor and mimic a microenvironment for cell proliferation and differentiation. According to previous studies, poly(lactic acid) and multi-wall carbon nanotubes have been introduced as novel and promising biomaterials for the proliferation and differentiation of stem cells. Some cell types have been successfully grown on a matrix containing carbon nanotubes in tissue engineering but there is no report for this material to support stem cells differentiation into germ cells lineage. This study designed a 3D wet-electrospun poly(lactic acid) and poly(lactic acid)/multi-wall carbon nanotubes composite scaffold to compare infiltration, proliferation, and differentiation potential of menstrual blood-derived stem cells toward germ cell lineage with 2D culture. Our primary data revealed that the fabricated scaffold has mechanical and biological suitable qualities for supporting and attachments of stem cells. The differentiated menstrual blood-derived stem cells tracking in scaffolds using scanning electron microscopy confirmed cell attachment, aggregation, and distribution on the porous scaffold. Based on the differentiation assay by RT-PCR analysis, stem cells and germ-like cells markers were expressed in 3D groups as well as 2D one. It seems that poly(lactic acid)/multi-wall carbon nanotubes scaffold-seeded menstrual blood-derived stem cells could be viewed as a novel, safe, and accessible construct for these cells, as they enhance germ-like generation from menstrual blood-derived stem cells.

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells.

PubMed

Zarà, Marta; Canobbio, Ilaria; Visconte, Caterina; Canino, Jessica; Torti, Mauro; Guidetti, Gianni Francesco

2018-08-01

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl 2 and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbβ3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca 2+ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A 2 synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP. Copyright © 2018 Elsevier Inc. All rights reserved.

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells

PubMed Central

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today. PMID:27362493

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells.

PubMed

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

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

1994-01-01

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

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

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

1997-01-01

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

Protein ubiquitination in postsynaptic densities after hypoxia in rat neostriatum is blocked by hypothermia.

PubMed

Capani, Francisco; Saraceno, Gustavo Ezequiel; Botti, Valeria; Aon-Bertolino, Laura; de Oliveira, Diêgo Madureira; Barreto, George; Galeano, Pablo; Giraldez-Alvarez, Lisandro Diego; Coirini, Héctor

2009-10-01

Synaptic dysfunction has been associated with neuronal cell death following hypoxia. The lack of knowledge on the mechanisms underlying this dysfunction prompted us to investigate the morphological changes in the postsynaptic densities (PSDs) induced by hypoxia. The results presented here demonstrate that PSDs of the rat neostriatum are highly modified and ubiquitinated 6 months after induction of hypoxia in a model of perinatal asphyxia. Using both two dimensional (2D) and three dimensional (3D) electron microscopic analyses of synapses stained with ethanolic phosphotungstic acid (E-PTA), we observed an increment of PSD thickness dependent on the duration and severity of the hypoxic insult. The PSDs showed clear signs of damage and intense staining for ubiquitin. These morphological and molecular changes were effectively blocked by hypothermia treatment, one of the most effective strategies for hypoxia-induced brain injury available today. Our data suggest that synaptic dysfunction following hypoxia may be caused by long-term misfolding and aggregation of proteins in the PSD.

Development of scaffold architectures and heterotypic cell systems for hepatocyte transplantation

NASA Astrophysics Data System (ADS)

Alzebdeh, Dalia Abdelrahim

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

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

PubMed

Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

2016-10-31

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Molecular insights into early stage aggregation of di-Fmoc-L-lysine in binary mixture of organic solvent and water

NASA Astrophysics Data System (ADS)

Huda, Md Masrul; Rai, Neeraj

Molecular gels are relatively new class of soft materials, which are formed by the supramolecular aggregation of low molecular weight gelators (LMWGs) in organic solvents and/or water. Hierarchical self-assembly of small gelator molecules lead to three-dimensional complex fibrillar networks, which restricts the flow of solvents and results in viscous solid like materials or gels. These gels have drawn significant attentions for their potential applications for drug delivery, tissue engineering, materials for sensors etc. As of now, self-assembly of gelator molecules into one-dimensional fibers is not well understood, although that is very important to design new gelators for desired applications. Here, we present molecular dynamics study that provides molecular level insight into early stage aggregation of selected gelator, di-Fmoc-L-lysine in binary mixture of organic solvent and water. We will present the role of different functional groups of gelator molecule such as aromatic ring, amide, and carboxylic group on aggregation. We will also present the effect of concentrations of gelator and solvent on self-assembly of gelators. This study has captured helical fiber growth and branching of fiber, which is in good agreement with experimental observations.

Three-dimensional finite elements for the analysis of soil contamination using a multiple-porosity approach

NASA Astrophysics Data System (ADS)

El-Zein, Abbas; Carter, John P.; Airey, David W.

2006-06-01

A three-dimensional finite-element model of contaminant migration in fissured clays or contaminated sand which includes multiple sources of non-equilibrium processes is proposed. The conceptual framework can accommodate a regular network of fissures in 1D, 2D or 3D and immobile solutions in the macro-pores of aggregated topsoils, as well as non-equilibrium sorption. A Galerkin weighted-residual statement for the three-dimensional form of the equations in the Laplace domain is formulated. Equations are discretized using linear and quadratic prism elements. The system of algebraic equations is solved in the Laplace domain and solution is inverted to the time domain numerically. The model is validated and its scope is illustrated through the analysis of three problems: a waste repository deeply buried in fissured clay, a storage tank leaking into sand and a sanitary landfill leaching into fissured clay over a sand aquifer.

Social aggregation in the pelagic zone with special reference to fish and invertebrates.

PubMed

Ritz, David A; Hobday, Alistair J; Montgomery, John C; Ward, Ashley J W

2011-01-01

Aggregations of organisms, ranging from zooplankton to whales, are an extremely common phenomenon in the pelagic zone; perhaps the best known are fish schools. Social aggregation is a special category that refers to groups that self-organize and maintain cohesion to exploit benefits such as protection from predators, and location and capture of resources more effectively and with greater energy efficiency than could a solitary individual. In this review we explore general aggregation principles, with specific reference to pelagic organisms; describe a range of new technologies either designed for studying aggregations or that could potentially be exploited for this purpose; report on the insights gained from theoretical modelling; discuss the relationship between social aggregation and ocean management; and speculate on the impact of climate change. Examples of aggregation occur in all animal phyla. Among pelagic organisms, it is possible that repeated co-occurrence of stable pairs of individuals, which has been established for some schooling fish, is the likely precursor leading to networks of social interaction and more complex social behaviour. Social network analysis has added new insights into social behaviour and allows us to dissect aggregations and to examine how the constituent individuals interact with each other. This type of analysis is well advanced in pinnipeds and cetaceans, and work on fish is progressing. Detailed three-dimensional analysis of schools has proved to be difficult, especially at sea, but there has been some progress recently. The technological aids for studying social aggregation include video and acoustics, and have benefited from advances in digitization, miniaturization, motion analysis and computing power. New techniques permit three-dimensional tracking of thousands of individual animals within a single group which has allowed novel insights to within-group interactions. Approaches using theoretical modelling of aggregations have a long history but only recently have hypotheses been tested empirically. The lack of synchrony between models and empirical data, and lack of a common framework to schooling models have hitherto hampered progress; however, recent developments in this field offer considerable promise. Further, we speculate that climate change, already having effects on ecosystems, could have dramatic effects on aggregations through its influence on species composition by altering distribution ranges, migration patterns, vertical migration, and oceanic acidity. Because most major commercial fishing targets schooling species, these changes could have important consequences for the dependent businesses. 2011 Elsevier Ltd. All rights reserved.

3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads.

PubMed

Lehmann, R; Gallert, C; Roddelkopf, T; Junginger, S; Wree, A; Thurow, K

2016-08-01

Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek(®) Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.

A new technique for calculating individual dermal fibroblast contractile forces generated within collagen-GAG scaffolds.

PubMed

Harley, Brendan A; Freyman, Toby M; Wong, Matthew Q; Gibson, Lorna J

2007-10-15

Cell-mediated contraction plays a critical role in many physiological and pathological processes, notably organized contraction during wound healing. Implantation of an appropriately formulated (i.e., mean pore size, chemical composition, degradation rate) three-dimensional scaffold into an in vivo wound site effectively blocks the majority of organized wound contraction and results in induced regeneration rather than scar formation. Improved understanding of cell contraction within three-dimensional constructs therefore represents an important area of study in tissue engineering. Studies of cell contraction within three-dimensional constructs typically calculate an average contractile force from the gross deformation of a macroscopic substrate by a large cell population. In this study, cellular solids theory has been applied to conventional column buckling relationships to quantify the magnitude of individual cell contraction events within a three-dimensional, collagen-glycosaminoglycan scaffold. This new technique can be used for studying cell mechanics with a wide variety of porous scaffolds that resemble low-density, open-cell foams. It extends previous methods for analyzing cell buckling of two-dimensional substrates to three-dimensional constructs. From data available in the literature, the mean contractile force (Fc) generated by individual dermal fibroblasts within the collagen-glycosaminoglycan scaffold was calculated to range between 11 and 41 nN (Fc=26+/-13 nN, mean+/-SD), with an upper bound of cell contractility estimated at 450 nN.

Multiple Attribute Group Decision-Making Methods Based on Trapezoidal Fuzzy Two-Dimensional Linguistic Partitioned Bonferroni Mean Aggregation Operators.

PubMed

Yin, Kedong; Yang, Benshuo; Li, Xuemei

2018-01-24

In this paper, we investigate multiple attribute group decision making (MAGDM) problems where decision makers represent their evaluation of alternatives by trapezoidal fuzzy two-dimensional uncertain linguistic variable. To begin with, we introduce the definition, properties, expectation, operational laws of trapezoidal fuzzy two-dimensional linguistic information. Then, to improve the accuracy of decision making in some case where there are a sort of interrelationship among the attributes, we analyze partition Bonferroni mean (PBM) operator in trapezoidal fuzzy two-dimensional variable environment and develop two operators: trapezoidal fuzzy two-dimensional linguistic partitioned Bonferroni mean (TF2DLPBM) aggregation operator and trapezoidal fuzzy two-dimensional linguistic weighted partitioned Bonferroni mean (TF2DLWPBM) aggregation operator. Furthermore, we develop a novel method to solve MAGDM problems based on TF2DLWPBM aggregation operator. Finally, a practical example is presented to illustrate the effectiveness of this method and analyses the impact of different parameters on the results of decision-making.

Multiple Attribute Group Decision-Making Methods Based on Trapezoidal Fuzzy Two-Dimensional Linguistic Partitioned Bonferroni Mean Aggregation Operators

PubMed Central

Yin, Kedong; Yang, Benshuo

2018-01-01

In this paper, we investigate multiple attribute group decision making (MAGDM) problems where decision makers represent their evaluation of alternatives by trapezoidal fuzzy two-dimensional uncertain linguistic variable. To begin with, we introduce the definition, properties, expectation, operational laws of trapezoidal fuzzy two-dimensional linguistic information. Then, to improve the accuracy of decision making in some case where there are a sort of interrelationship among the attributes, we analyze partition Bonferroni mean (PBM) operator in trapezoidal fuzzy two-dimensional variable environment and develop two operators: trapezoidal fuzzy two-dimensional linguistic partitioned Bonferroni mean (TF2DLPBM) aggregation operator and trapezoidal fuzzy two-dimensional linguistic weighted partitioned Bonferroni mean (TF2DLWPBM) aggregation operator. Furthermore, we develop a novel method to solve MAGDM problems based on TF2DLWPBM aggregation operator. Finally, a practical example is presented to illustrate the effectiveness of this method and analyses the impact of different parameters on the results of decision-making. PMID:29364849

An Energy Model of Place Cell Network in Three Dimensional Space.

PubMed

Wang, Yihong; Xu, Xuying; Wang, Rubin

2018-01-01

Place cells are important elements in the spatial representation system of the brain. A considerable amount of experimental data and classical models are achieved in this area. However, an important question has not been addressed, which is how the three dimensional space is represented by the place cells. This question is preliminarily surveyed by energy coding method in this research. Energy coding method argues that neural information can be expressed by neural energy and it is convenient to model and compute for neural systems due to the global and linearly addable properties of neural energy. Nevertheless, the models of functional neural networks based on energy coding method have not been established. In this work, we construct a place cell network model to represent three dimensional space on an energy level. Then we define the place field and place field center and test the locating performance in three dimensional space. The results imply that the model successfully simulates the basic properties of place cells. The individual place cell obtains unique spatial selectivity. The place fields in three dimensional space vary in size and energy consumption. Furthermore, the locating error is limited to a certain level and the simulated place field agrees to the experimental results. In conclusion, this is an effective model to represent three dimensional space by energy method. The research verifies the energy efficiency principle of the brain during the neural coding for three dimensional spatial information. It is the first step to complete the three dimensional spatial representing system of the brain, and helps us further understand how the energy efficiency principle directs the locating, navigating, and path planning function of the brain.

A physical description of the adhesion and aggregation of platelets

NASA Astrophysics Data System (ADS)

Chopard, Bastien; de Sousa, Daniel Ribeiro; Lätt, Jonas; Mountrakis, Lampros; Dubois, Frank; Yourassowsky, Catherine; Van Antwerpen, Pierre; Eker, Omer; Vanhamme, Luc; Perez-Morga, David; Courbebaisse, Guy; Lorenz, Eric; Hoekstra, Alfons G.; Boudjeltia, Karim Zouaoui

2017-04-01

The early stages of clot formation in blood vessels involve platelet adhesion-aggregation. Although these mechanisms have been extensively studied, gaps in their understanding still persist. We have performed detailed in vitro experiments, using the well-known Impact-R device, and developed a numerical model to better describe and understand this phenomenon. Unlike previous studies, we took into account the differential role of pre-activated and non-activated platelets, as well as the three-dimensional nature of the aggregation process. Our investigation reveals that blood albumin is a major parameter limiting platelet aggregate formation in our experiment. Simulations are in very good agreement with observations and provide quantitative estimates of the adhesion and aggregation rates that are hard to measure experimentally. They also provide a value of the effective diffusion of platelets in blood subject to the shear rate produced by the Impact-R.

Regeneration of dental pulp/dentine complex with a three-dimensional and scaffold-free stem-cell sheet-derived pellet.

PubMed

Na, Sijia; Zhang, Hao; Huang, Fang; Wang, Weiqi; Ding, Yin; Li, Dechao; Jin, Yan

2016-03-01

Dental pulp/dentine complex regeneration is indispensable to the construction of biotissue-engineered tooth roots and represents a promising approach to therapy for irreversible pulpitis. We used a tissue-engineering method based on odontogenic stem cells to design a three-dimensional (3D) and scaffold-free stem-cell sheet-derived pellet (CSDP) with the necessary physical and biological properties. Stem cells were isolated and identified and stem cells from root apical papilla (SCAPs)-based CSDPs were then fabricated and examined. Compact cell aggregates containing a high proportion of extracellular matrix (ECM) components were observed, and the CSDP culture time was prolonged. The expression of alkaline phosphatase (ALP), dentine sialoprotein (DSPP), bone sialoprotein (BSP) and runt-related gene 2 (RUNX2) mRNA was higher in CSDPs than in cell sheets (CSs), indicating that CSDPs have greater odonto/osteogenic potential. To further investigate this hypothesis, CSDPs and CSs were inserted into human treated dentine matrix fragments (hTDMFs) and transplanted into the subcutaneous space in the backs of immunodeficient mice, where they were cultured in vivo for 6 weeks. The root space with CSDPs was filled entirely with a dental pulp-like tissue with well-established vascularity, and a continuous layer of dentine-like tissue was deposited onto the existing dentine. A layer of odontoblast-like cells was found to express DSPP, ALP and BSP, and human mitochondria lined the surface of the newly formed dentine-like tissue. These results clearly indicate that SCAP-CSDPs with a mount of endogenous ECM have a strong capacity to form a heterotopic dental pulp/dentine complex in empty root canals; this method can be used in the fabrication of bioengineered dental roots and also provides an alternative treatment approach for pulp disease. Copyright © 2013 John Wiley & Sons, Ltd.

Toward a 3D model of human brain development for studying gene/environment interactions

PubMed Central

2013-01-01

This project aims to establish and characterize an in vitro model of the developing human brain for the purpose of testing drugs and chemicals. To accurately assess risk, a model needs to recapitulate the complex interactions between different types of glial cells and neurons in a three-dimensional platform. Moreover, human cells are preferred over cells from rodents to eliminate cross-species differences in sensitivity to chemicals. Previously, we established conditions to culture rat primary cells as three-dimensional aggregates, which will be humanized and evaluated here with induced pluripotent stem cells (iPSCs). The use of iPSCs allows us to address gene/environment interactions as well as the potential of chemicals to interfere with epigenetic mechanisms. Additionally, iPSCs afford us the opportunity to study the effect of chemicals during very early stages of brain development. It is well recognized that assays for testing toxicity in the developing brain must consider differences in sensitivity and susceptibility that arise depending on the time of exposure. This model will reflect critical developmental processes such as proliferation, differentiation, lineage specification, migration, axonal growth, dendritic arborization and synaptogenesis, which will probably display differences in sensitivity to different types of chemicals. Functional endpoints will evaluate the complex cell-to-cell interactions that are affected in neurodevelopment through chemical perturbation, and the efficacy of drug intervention to prevent or reverse phenotypes. The model described is designed to assess developmental neurotoxicity effects on unique processes occurring during human brain development by leveraging human iPSCs from diverse genetic backgrounds, which can be differentiated into different cell types of the central nervous system. Our goal is to demonstrate the feasibility of the personalized model using iPSCs derived from individuals with neurodevelopmental disorders caused by known mutations and chromosomal aberrations. Notably, such a human brain model will be a versatile tool for more complex testing platforms and strategies as well as research into central nervous system physiology and pathology. PMID:24564953

Repressive effects of oat extracts on intracellular lipid-droplet formation in adipocytes and a three-dimensional subcutaneous adipose tissue model.

PubMed

Kato, Shinya; Kato, Yuko; Shibata, Hiroki; Saitoh, Yasukazu; Miwa, Nobuhiko

2015-04-01

We assessed the repression of lipid-droplet formation in mouse mesenchymal stromal preadipocytes OP9 by specified oat extracts (Hatomugi, Coix lacryma-jobi var. ma-yuen) named "SPH" which were proteolytically and glucosyl-transferredly prepared from finely-milled oat whole-grain. Stimulation of OP9 preadipocytes with insulin-containing serum-replacement promoted differentiation to adipocytes, concurrently with an increase in the intracellular lipid droplets by 51.5%, which were repressed by SPH-bulk or SPH-water-extract at 840ppm, to 33.5% or 46.9%, respectively, but not by SPH-ethanol-extract at the same dose, showing the hydrophilic property of the anti-adipogenetic ingredients. The intracellular lipid droplets were scanty for intact preadipocytes, small-sized but abundant for the SPH-unadministered adipocytes, and large-sized but few for SPH-bulk-administered adipocytes being coexistent with many lipid-droplet-lacking viable cells, suggesting "the all-or-none rule" for lipid-droplet generation in cell-to-cell. Hydrogen-peroxide-induced cell death in human epidermal keratinocytes HaCaT was prevented by SPH-bulk at 100 or 150ppm by 5.6-8.1%, being consistent with higher viabilities of SPH-bulk-administered OP9 cells, together with repressions of both cell shrinkage and cell detachment from the culture substratum. In three-dimensional subcutaneous adipose tissue models reconstructed with HaCaT-keratinocytes and OP9-preadipocytes, lipid droplets were accumulated in dermal OP9-cell-parts, and repressed to 43.5% by SPH-bulk at 840ppm concurrently with marked diminishment of huge aggregates of lipid droplets. Thus SPH-bulk suppresses adipogenesis-associated lipid-droplet accumulation during differentiation of OP9 preadipocytes together with lowered cytotoxicity to either HaCaT keratinocytes or the preadipocytes. Copyright © 2015 Elsevier B.V. All rights reserved.

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

PubMed

Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

2012-03-01

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

GEOMETRIC CROSS SECTIONS OF DUST AGGREGATES AND A COMPRESSION MODEL FOR AGGREGATE COLLISIONS

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

Suyama, Toru; Wada, Koji; Tanaka, Hidekazu

2012-07-10

Geometric cross sections of dust aggregates determine their coupling with disk gas, which governs their motions in protoplanetary disks. Collisional outcomes also depend on geometric cross sections of initial aggregates. In a previous paper, we performed three-dimensional N-body simulations of sequential collisions of aggregates composed of a number of sub-micron-sized icy particles and examined radii of gyration (and bulk densities) of the obtained aggregates. We showed that collisional compression of aggregates is not efficient and that aggregates remain fluffy. In the present study, we examine geometric cross sections of the aggregates. Their cross sections decrease due to compression as wellmore » as to their gyration radii. It is found that a relation between the cross section and the gyration radius proposed by Okuzumi et al. is valid for the compressed aggregates. We also refine the compression model proposed in our previous paper. The refined model enables us to calculate the evolution of both gyration radii and cross sections of growing aggregates and reproduces well our numerical results of sequential aggregate collisions. The refined model can describe non-equal-mass collisions as well as equal-mass cases. Although we do not take into account oblique collisions in the present study, oblique collisions would further hinder compression of aggregates.« less

Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc.

PubMed

Gruber, H E; Hanley, E N

2009-06-01

The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.

Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Spaulding, Glenn F.; Tsao, Yow-Min D.; Flechsig, Scott; Jones, Leslie; Soehnge, Holly

2003-01-01

The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissuelike assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities. The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding.

PubMed

Kannan, R; Labotka, R; Low, P S

1988-09-25

Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.

Cadherin Composition and Multicellular Aggregate Invasion In Organotypic Models of Epithelial Ovarian Cancer Intraperitoneal Metastasis

PubMed Central

Klymenko, Yuliya; Kim, Oleg; Loughran, Elizabeth; Yang, Jing; Lombard, Rachel; Alber, Mark; Stack, M. Sharon

2017-01-01

During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multi-cellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich sub-mesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior, however the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the sub-mesothelial collagen matrix. Acquisition of Ncad by E-cadherin expressing cells (Ecad+) increased mesothelial clearance activity, but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a “leader-follower” mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad+ in pre-clinical models of EOC metastasis. PMID:28628116

Cadherin composition and multicellular aggregate invasion in organotypic models of epithelial ovarian cancer intraperitoneal metastasis.

PubMed

Klymenko, Y; Kim, O; Loughran, E; Yang, J; Lombard, R; Alber, M; Stack, M S

2017-10-19

During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.

Determinants of platelet aggregation in 50-70-year-old men from three Japanese communities.

PubMed

Imano, Hironori; Iso, Hiroyasu; Sato, Shinichi; Kitamura, Akihiko; Okamura, Tomonori; Tanigawa, Takeshi; Ohira, Tetsuya; Kudo, Minako; Naito, Yoshihiko; Iida, Minoru; Shimamoto, Takashi

2002-12-01

To investigate the association of lifestyle and constitutional variables with platelet aggregation, we examined the platelet aggregation, serum fatty acid composition, alcohol intake, smoking, and dietary intake of seafood and soybean estimated by a 1-week dietary record in 448 males aged 50-70 in three rural Japanese communities: Ikawa, Akita prefecture (northeast coast), Noichi, Kochi prefecture (southwest coast), and Kyowa, Ibaraki prefecture (central inland). Platelet aggregatory threshold index (PATI) was used to determine the minimum concentration of adenosine 5'-diphosphate (ADP) that caused a non-reversible aggregation of platelets. Intake of seafood and n3-polyunsaturated fatty acid and ingestion of ethanol were higher in the northeast coastal community than in the other two communities. Mean platelet and white blood cell counts were lower in northeast coastal community than in the other two communities. The geometric mean PATI was higher (i.e. platelet aggregation was lower) in the northeast coastal community than the other two communities. Within the entire sample, platelet aggregation correlated inversely with serum level of n3-polyunsaturated fatty acids and gamma-glutamyl transpeptidase, an index of alcohol consumption, and positively with platelet and white blood cell counts. Platelet aggregation tended to correlate positively with serum arachidonic acid. There was no correlation between smoking and platelet aggregation. Our results suggest that seafood intake and moderate alcohol consumption reduce platelet aggregation.

Aggregation and Disaggregation of Senile Plaques in Alzheimer Disease

NASA Astrophysics Data System (ADS)

Cruz, L.; Urbanc, B.; Buldyrev, S. V.; Christie, R.; Gomez-Isla, T.; Havlin, S.; McNamara, M.; Stanley, H. E.; Hyman, B. T.

1997-07-01

We quantitatively analyzed, using laser scanning confocal microscopy, the three-dimensional structure of individual senile plaques in Alzheimer disease. We carried out the quantitative analysis using statistical methods to gain insights about the processes that govern Aβ peptide deposition. Our results show that plaques are complex porous structures with characteristic pore sizes. We interpret plaque morphology in the context of a new dynamical model based on competing aggregation and disaggregation processes in kinetic steady-state equilibrium with an additional diffusion process allowing Aβ deposits to diffuse over the surface of plaques.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

PubMed Central

2012-01-01

Introduction Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. Methods Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. Results Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. Conclusions Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment. PMID:22676383

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

PubMed Central

Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

2011-01-01

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

Three-dimensional behavior of ice crystals and biological cells during freezing of cell suspensions.

PubMed

Ishiguro, H; Koike, K

1998-09-11

Behavior of ice crystals and human red blood cells during extracellular-freezing was investigated in three-dimensions using a confocal laser scanning microscope(CLSM), which noninvasively produces tomograms of biological materials. Physiological saline and physiological saline with 2.4 M glycerol were used for suspension. Various cooling rates for directional solidification were used for distinctive morphology of the ice crystals. Addition of acridine orange as a fluorescent dye into the cell suspension enabled ice crystal, cells and unfrozen solution to be distinguished by different colors. The results indicate that the microscopic structure is three-dimensional for flat, cellular, and dendritic solid-liquid interfaces and that a CLSM is very effective in studying three-dimensional structure during the freezing of cell suspensions.

Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

PubMed

Murphy, Kaitlin C; Whitehead, Jacklyn; Falahee, Patrick C; Zhou, Dejie; Simon, Scott I; Leach, J Kent

2017-06-01

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E 2 (PGE 2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE 2 and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE 2 and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells 2017;35:1493-1504. © 2017 AlphaMed Press.

From particle condensation to polymer aggregation

NASA Astrophysics Data System (ADS)

Janke, Wolfhard; Zierenberg, Johannes

2018-01-01

We draw an analogy between droplet formation in dilute particle and polymer systems. Our arguments are based on finite-size scaling results from studies of a two-dimensional lattice gas to three-dimensional bead-spring polymers. To set the results in perspective, we compare with in part rigorous theoretical scaling laws for canonical condensation in a supersaturated gas at fixed temperature, and derive corresponding scaling predictions for an undercooled gas at fixed density. The latter allows one to efficiently employ parallel multicanonical simulations and to reach previously not accessible scaling regimes. While the asymptotic scaling can not be observed for the comparably small polymer system sizes, they demonstrate an intermediate scaling regime also observable for particle condensation. Altogether, our extensive results from computer simulations provide clear evidence for the close analogy between particle condensation and polymer aggregation in dilute systems.

Long-Term Results of Cartilage Repair after Allogeneic Transplantation of Cartilaginous Aggregates Formed from Bone Marrow–Derived Cells for Large Osteochondral Defects in Rabbit Knees

PubMed Central

Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa

2013-01-01

Objective: The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow–derived cells. Methods: Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow–derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O’Driscoll score). Results: The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O’Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. Conclusions: This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow–derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated. PMID:26069678

Three-Dimensional Gene Map of Cancer Cell Types: Structural Entropy Minimisation Principle for Defining Tumour Subtypes

PubMed Central

Li, Angsheng; Yin, Xianchen; Pan, Yicheng

2016-01-01

In this study, we propose a method for constructing cell sample networks from gene expression profiles, and a structural entropy minimisation principle for detecting natural structure of networks and for identifying cancer cell subtypes. Our method establishes a three-dimensional gene map of cancer cell types and subtypes. The identified subtypes are defined by a unique gene expression pattern, and a three-dimensional gene map is established by defining the unique gene expression pattern for each identified subtype for cancers, including acute leukaemia, lymphoma, multi-tissue, lung cancer and healthy tissue. Our three-dimensional gene map demonstrates that a true tumour type may be divided into subtypes, each defined by a unique gene expression pattern. Clinical data analyses demonstrate that most cell samples of an identified subtype share similar survival times, survival indicators and International Prognostic Index (IPI) scores and indicate that distinct subtypes identified by our algorithms exhibit different overall survival times, survival ratios and IPI scores. Our three-dimensional gene map establishes a high-definition, one-to-one map between the biologically and medically meaningful tumour subtypes and the gene expression patterns, and identifies remarkable cells that form singleton submodules. PMID:26842724

Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

PubMed Central

Coquel, Anne-Sophie; Jacob, Jean-Pascal; Primet, Mael; Demarez, Alice; Dimiccoli, Mariella; Julou, Thomas; Moisan, Lionel

2013-01-01

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli. PMID:23633942

Exopolysaccharide microchannels direct bacterial motility and organize multicellular behavior

DOE PAGES

Berleman, James E.; Zemla, Marcin; Remis, Jonathan P.; ...

2016-05-06

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required formore » cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS - mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus.« less

Role of cellular adhesions in tissue dynamics spectroscopy

NASA Astrophysics Data System (ADS)

Merrill, Daniel A.; An, Ran; Turek, John; Nolte, David

2014-02-01

Cellular adhesions play a critical role in cell behavior, and modified expression of cellular adhesion compounds has been linked to various cancers. We tested the role of cellular adhesions in drug response by studying three cellular culture models: three-dimensional tumor spheroids with well-developed cellular adhesions and extracellular matrix (ECM), dense three-dimensional cell pellets with moderate numbers of adhesions, and dilute three-dimensional cell suspensions in agarose having few adhesions. Our technique for measuring the drug response for the spheroids and cell pellets was biodynamic imaging (BDI), and for the suspensions was quasi-elastic light scattering (QELS). We tested several cytoskeletal chemotherapeutic drugs (nocodazole, cytochalasin-D, paclitaxel, and colchicine) on three cancer cell lines chosen from human colorectal adenocarcinoma (HT-29), human pancreatic carcinoma (MIA PaCa-2), and rat osteosarcoma (UMR-106) to exhibit differences in adhesion strength. Comparing tumor spheroid behavior to that of cell suspensions showed shifts in the spectral motion of the cancer tissues that match predictions based on different degrees of cell-cell contacts. The HT-29 cell line, which has the strongest adhesions in the spheroid model, exhibits anomalous behavior in some cases. These results highlight the importance of using three-dimensional tissue models in drug screening with cellular adhesions being a contributory factor in phenotypic differences between the drug responses of tissue and cells.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

2005-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer

Edible oil structures at low and intermediate concentrations. II. Ultra-small angle X-ray scattering of in situ tristearin solids in triolein

NASA Astrophysics Data System (ADS)

Peyronel, Fernanda; Ilavsky, Jan; Mazzanti, Gianfranco; Marangoni, Alejandro G.; Pink, David A.

2013-12-01

Ultra-small angle X-ray scattering has been used for the first time to elucidate, in situ, the aggregation structure of a model edible oil system. The three-dimensional nano- to micro-structure of tristearin solid particles in triolein solvent was investigated using 5, 10, 15, and 20% solids. Three different sample preparation procedures were investigated: two slow cooling rates of 0.5°/min, case 1 (22 days of storage at room temperature) and case 2 (no storage), and one fast cooling of 30°/min, case 3 (no storage). The length scale investigated, by using the Bonse-Hart camera at beamline ID-15D at the Advanced Photon Source, Argonne National Laboratory, covered the range from 300 Å to 10 μm. The unified fit and the Guinier-Porod models in the Irena software were used to fit the data. The former was used to fit 3 structural levels. Level 1 structures showed that the primary scatterers were essentially 2-dimensional objects for the three cases. The scatterers possessed lateral dimensions between 1000 and 4300 Å. This is consistent with the sizes of crystalline nanoplatelets present which were observed using cryo-TEM. Level 2 structures were aggregates possessing radii of gyration, Rg2 between 1800 Å and 12000 Å and fractal dimensions of either D2=1 for case 3 or 1.8≤D2≤2.1 for case 1 and case 2. D2 = 1 is consistent with unaggregated 1-dimensional objects. 1.8 ≤ D2 ≤ 2.1 is consistent with these 1-dimensional objects (below) forming structures characteristic of diffusion or reaction limited cluster-cluster aggregation. Level 3 structures showed that the spatial distribution of the level 2 structures was uniform, on the average, for case 1, with fractal dimension D3≈3 while for case 2 and case 3 the fractal dimension was D3≈2.2, which suggested that the large-scale distribution had not come to equilibrium. The Guinier-Porod model showed that the structures giving rise to the aggregates with a fractal dimension given by D2 in the unified fit level 2 model were cylinders described by the parameter s ≈1 in the Guinier-Porod model. The size of the base of these cylinders was in agreement with the cryo-TEM observations as well as with the results of the level 1 unified fit model. By estimating the size of the nanoplatelets and understanding the structures formed via their aggregation, it will be possible to engineer novel lipids systems that embody desired functional characteristics.

Three-dimensional cell to tissue development process

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Parker, Clayton R. (Inventor)

2008-01-01

An improved three-dimensional cell to tissue development process using a specific time varying electromagnetic force, pulsed, square wave, with minimum fluid shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region.

Learning the Cell Structures with Three-Dimensional Models: Students' Achievement by Methods, Type of School and Questions' Cognitive Level

NASA Astrophysics Data System (ADS)

Lazarowitz, Reuven; Naim, Raphael

2013-08-01

The cell topic was taught to 9th-grade students in three modes of instruction: (a) students "hands-on," who constructed three-dimensional cell organelles and macromolecules during the learning process; (b) teacher demonstration of the three-dimensional model of the cell structures; and (c) teaching the cell topic with the regular learning material in an expository mode (which use one- or two-dimensional cell structures as are presented in charts, textbooks and microscopic slides). The sample included 669, 9th-grade students from 25 classes who were taught by 22 Biology teachers. Students were randomly assigned to the three modes of instruction, and two tests in content knowledge in Biology were used. Data were treated with multiple analyses of variance. The results indicate that entry behavior in Biology was equal for all the study groups and types of schools. The "hands-on" learning group who build three-dimensional models through the learning process achieved significantly higher on academic achievements and on the high and low cognitive questions' levels than the other two groups. The study indicates the advantages students may have being actively engaged in the learning process through the "hands-on" mode of instruction/learning.

Effect of chitosan conduit under a dynamic culture on the proliferation and neural differentiation of human exfoliated deciduous teeth stem cells.

PubMed

Su, Wen-Ta; Shih, Yi-An; Ko, Chih-Sheng

2016-06-01

Ex vivo engineering of artificial nerve conduit is a suitable alternative clinical treatment for nerve injuries. Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. These cells, when cultured in six-well plates, exhibited a spindle fibroblastic morphology, whereas those under a dynamic culture aggregated into neurosphere-like clusters in the chitosan conduit. In this study, we confirmed that SHEDs efficiently express the neural stem cell marker nestin, the early neural cell marker β-III-tubulin, the late neural marker neuron-specific enolase and the glial cell markers glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). The three-dimensional chitosan conduit and dynamic culture system generated fluid shear stress and enhanced nutrient transfer, promoting the differentiation of SHEDs to neural cells. In particular, the gene expressions of GFAP and CNPase increased by 28- and 53-fold, respectively. This study provides evidence for the dynamic culture of SHEDs during ex vivo neural differentiation and demonstrates its potential for cell therapy in neurological diseases. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Micro-Masonry: Construction of 3D Structures by Mesoscale Self-Assembly

PubMed Central

Fernandez, Javier G.; Khademhosseini, Ali

2010-01-01

A general method for construction of three dimensional structures by directed assembly of microscale polymeric sub-units is presented. Shape-controlled microgels are directed to assemble into different shapes by limiting their movement onto a molded substrate. The capillary forces, resulting from the presence of a liquid polymer, assemble the microgels in close contact with the rest of the units and with the free surface, the latter imposing the final geometry of the resulting construct. The result is a freestanding structure composed of one or multiple layers of sub-units assembled in a tightly packed conformation. The applicability of the technique for the construction of scaffolds with cell-laden sub-units is demonstrated. In addition, scaffolds formed by the sequential aggregation of sub-units are produced. PMID:20440697

Three-dimensional epithelial tissues generated from human embryonic stem cells.

PubMed

Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

2009-11-01

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

Improving the quality of miniature pig somatic cell nuclear transfer blastocysts: aggregation of SCNT embryos at the four-cell stage.

PubMed

Terashita, Y; Sugimura, S; Kudo, Y; Amano, R; Hiradate, Y; Sato, E

2011-04-01

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. © 2010 Blackwell Verlag GmbH.

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease.

PubMed

Mignot, Cyril; Delarasse, Cécile; Escaich, Séverine; Della Gaspera, Bruno; Noé, Eric; Colucci-Guyon, Emma; Babinet, Charles; Pekny, Milos; Vicart, Patrick; Boespflug-Tanguy, Odile; Dautigny, André; Rodriguez, Diana; Pham-Dinh, Danielle

2007-08-01

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

Structural, Optical, and Photocatalytic Properties of Quasi-One-Dimensional Nanocrystalline ZnO, ZnOC:nC Composites, and C-doped ZnO

NASA Astrophysics Data System (ADS)

Shalaeva, E. V.; Gyrdasova, O. I.; Krasilnikov, V. N.; Melkozerova, M. A.; Baklanova, I. V.; Buldakova, L. Yu.

Various thermolysis rotes of zinc glicolate complexes are considered for the synthesis of quasi-one-dimensional nanostructured aggregates ZnO and Zn-O-C used as photocatalysts. Structural features of quasi-one-dimensional aggregates Zn-O-C and ZnO are investigated in detail. Transmission electron microscopy, Raman spectroscopy, and electron paramagnetic resonance spectroscopy methods demonstrate that the aggregates Zn-O-C have either composite structure (ZnO crystallites in amorphous carbon matrix) or a C-doped ZnO single-phase structure depending on heat treatment conditions, and that all the aggregates exhibit as a rule a tubular morphology, a nanocrystalline structure with a high specific surface area, and a high concentration of singly charged oxygen vacancies. The mechanism of the nanocrystalline structure formation is discussed and the effect of thermolysis condition on the formation of the textured structure of aggregates is investigated. The results of examination of the photocatalytic and optical absorption properties of the synthesized aggregates are presented. The photocatalytic activity for the hydroquinone oxidation reaction under ultraviolet and visible light increases in the series: the reference ZnO powder, quasi-one-dimensional ZnO, quasi-one-dimensional aggregates C-doped ZnO, and this tendency correlates with the reduction of the optical gap width. As a result of our studies, we have arrived at an important conclusion that thermal treatment of ZnO:nC composites allows a C-doped ZnO with high catalytic activity. This increasing photoactivity of C-doped ZnO aggregates is attributed to the optimal specific surface area and electron-energy spectrum restructuring to be produced owing to the presence of singly charged oxygen vacancies and carbon dissolved in the ZnO lattice.

SEROLOGICAL ANALYSES OF CELLULAR SLIME-MOLD DEVELOPMENT I.

PubMed Central

Sonneborn, D. R.; Sussman, M.; Levine, L.

1964-01-01

Sonneborn, D. R. (Brandeis University, Waltham, Mass.), M. Sussman, and L. Levine. Serological analysis of cellular slime-mold development. I. Changes in antigenic activity during cell aggregation. J. Bacteriol. 87:1321–1329. 1964.—During aggregation in Dictyostelium discoideum, the concentration of a single antigenic determinant increased markedly, starting from very low or undetectable levels. Subsequently, the determinant appeared to segregate preferentially into the stalks of terminal fruiting bodies. Sera containing the antibody specific for this determinant inhibited the aggregation of D. discoideum without disturbing cell viability. The properties of the antigen during fractionation are consistent with the supposition that it may be a protein associated with the cell membrane. The ability or inability of three species to coaggregate with D. discoideum was correlated with the presence or absence of the antigenic determinant in aggregates of these species. PMID:14188709

Hippocampal place-cell firing during movement in three-dimensional space

NASA Technical Reports Server (NTRS)

Knierim, J. J.; McNaughton, B. L.

2001-01-01

"Place" cells of the rat hippocampus are coupled to "head direction" cells of the thalamus and limbic cortex. Head direction cells are sensitive to head direction in the horizontal plane only, which leads to the question of whether place cells similarly encode locations in the horizontal plane only, ignoring the z axis, or whether they encode locations in three dimensions. This question was addressed by recording from ensembles of CA1 pyramidal cells while rats traversed a rectangular track that could be tilted and rotated to different three-dimensional orientations. Cells were analyzed to determine whether their firing was bound to the external, three-dimensional cues of the environment, to the two-dimensional rectangular surface, or to some combination of these cues. Tilting the track 45 degrees generally provoked a partial remapping of the rectangular surface in that some cells maintained their place fields, whereas other cells either gained new place fields, lost existing fields, or changed their firing locations arbitrarily. When the tilted track was rotated relative to the distal landmarks, most place fields remapped, but a number of cells maintained the same place field relative to the x-y coordinate frame of the laboratory, ignoring the z axis. No more cells were bound to the local reference frame of the recording apparatus than would be predicted by chance. The partial remapping demonstrated that the place cell system was sensitive to the three-dimensional manipulations of the recording apparatus. Nonetheless the results were not consistent with an explicit three-dimensional tuning of individual hippocampal neurons nor were they consistent with a model in which different sets of cells are tightly coupled to different sets of environmental cues. The results are most consistent with the statement that hippocampal neurons can change their "tuning functions" in arbitrary ways when features of the sensory input or behavioral context are altered. Understanding the rules that govern the remapping phenomenon holds promise for deciphering the neural circuitry underlying hippocampal function.

Identifying microbial habitats in soil using quantum dots and x-ray fluorescence microtomography

NASA Astrophysics Data System (ADS)

O'Brien, S. L.; Whiteside, M. D.; Sholto-Douglas, D.; Dohnalkova, A.; Durall, D. M.; Gursoy, D.; Jones, M. D.; Kovarik, L.; Lai, B.; Roehrig, C.; Sullivan, S.; Vogt, S.; Kemner, K. M.

2015-12-01

The metabolic activities of soil microbes are the primary drivers of biogeochemical processes controlling the terrestrial carbon cycle, nutrient availability to plants, contaminant remediation, water quality, and other ecosystem services. However, we have a limited understanding of microbial metabolic processes such as nutrient uptake rates, substrate preferences, or how microbes and microbial metabolism are distributed throughout the three-dimensional pore network of the soil. Here we use a novel combination of imaging techniques with quantum dots (QDs, engineered semiconductor nanoparticles that produce size or composition-dependent fluorescence) to locate bacteria in the three-dimensional pore network of a soil aggregate. First, we show using confocal and aberration-corrected transmission electron microscopies that bacteria (Bacillus subtilis, Pseudomonas fluorescens, and Pseudomonas protogens) actively take up and internalize CdSe/ZnS core/shell QDs conjugated to biologically relevant substrates. Next, we show that cells bearing QDs can be identified using fluorescence imaging with hard x-rays at 2ID-D at the Advanced Photon Source (APS). Finally, we demonstrate that the Se constituent to the QDs can be used to label bacteria in three-dimensional tomographic reconstructions of natural soil at 0.5 nm spatial resolution using hard x-rays at 2ID-E at the APS. This is the first time soil bacteria have been imaged in the intact soil matrix at such high resolution. These results offer a new way to experimentally investigate basic bacterial ecology in situ, revealing constraints on microbial function in soil that will help improve connections between pore-scale and ecosystem-scale processes in models.

Bistable Expression of CsgD in Salmonella enterica Serovar Typhimurium Connects Virulence to Persistence

PubMed Central

MacKenzie, Keith D.; Wang, Yejun; Shivak, Dylan J.; Wong, Cynthia S.; Hoffman, Leia J. L.; Lam, Shirley; Kröger, Carsten; Cameron, Andrew D. S.; Townsend, Hugh G. G.; Köster, Wolfgang

2015-01-01

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment. PMID:25824832

Predicting human blood viscosity in silico

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

Fedosov, Dmitry A.; Pan, Wenxiao; Caswell, Bruce

2011-07-05

Cellular suspensions such as blood are a part of living organisms and their rheological and flow characteristics determine and affect majority of vital functions. The rheological and flow properties of cell suspensions are determined by collective dynamics of cells, their structure or arrangement, cell properties and interactions. We study these relations for blood in silico using a mesoscopic particle-based method and two different models (multi-scale/low-dimensional) of red blood cells. The models yield accurate quantitative predictions of the dependence of blood viscosity on shear rate and hematocrit. We explicitly model cell aggregation interactions and demonstrate the formation of reversible rouleaux structuresmore » resulting in a tremendous increase of blood viscosity at low shear rates and yield stress, in agreement with experiments. The non-Newtonian behavior of such cell suspensions (e.g., shear thinning, yield stress) is analyzed and related to the suspension’s microstructure, deformation and dynamics of single cells. We provide the flrst quantitative estimates of normal stress differences and magnitude of aggregation forces in blood. Finally, the flexibility of the cell models allows them to be employed for quantitative analysis of a much wider class of complex fluids including cell, capsule, and vesicle suspensions.« less

Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks

PubMed Central

1981-01-01

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X- 100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed. PMID:6799521

Hydrogel-based three-dimensional cell culture for organ-on-a-chip applications.

PubMed

Lee, Seung Hwan; Shim, Kyu Young; Kim, Bumsang; Sung, Jong Hwan

2017-05-01

Recent studies have reported that three-dimensionally cultured cells have more physiologically relevant functions than two-dimensionally cultured cells. Cells are three-dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ-on-a-chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation-based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ-on-a-chip. Last, we also discuss the challenges of hydrogel-based three-dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580-589, 2017. © 2017 American Institute of Chemical Engineers.

[Advances in the research of application of hydrogels in three-dimensional bioprinting].

PubMed

Yang, J; Zhao, Y; Li, H H; Zhu, S H

2016-08-20

Hydrogels are three-dimensional networks made of hydrophilic polymer crosslinked through covalent bonds or physical intermolecular attractions, which can contain growth media and growth factors to support cell growth. In bioprinting, hydrogels are used to provide accurate control over cellular microenvironment and to dramatically reduce experimental repetition times, meanwhile we can obtain three-dimensional cell images of high quality. Hydrogels in three-dimensional bioprinting have received a considerable interest due to their structural similarities to the natural extracellular matrix and polyporous frameworks which can support the cellular proliferation and survival. Meanwhile, they are accompanied by many challenges.

A facile in vitro model to study rapid mineralization in bone tissues.

PubMed

Deegan, Anthony J; Aydin, Halil M; Hu, Bin; Konduru, Sandeep; Kuiper, Jan Herman; Yang, Ying

2014-09-16

Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required. We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT). Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture. This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.

Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions☆

PubMed Central

Soares, Filipa A.C.; Chandra, Amit; Thomas, Robert J.; Pedersen, Roger A.; Vallier, Ludovic; Williams, David J.

2014-01-01

The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes. PMID:24440272

Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

PubMed

Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

2015-04-01

The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture. © 2015 Wiley Periodicals, Inc.

A Novel Magnetic Actuation Scheme to Disaggregate Nanoparticles and Enhance Passage across the Blood–Brain Barrier

PubMed Central

Le, Tuan-Anh; Amin, Faiz Ul; Kim, Myeong Ok

2017-01-01

The blood–brain barrier (BBB) hinders drug delivery to the brain. Despite various efforts to develop preprogramed actuation schemes for magnetic drug delivery, the unmodeled aggregation phenomenon limits drug delivery performance. This paper proposes a novel scheme with an aggregation model for a feed-forward magnetic actuation design. A simulation platform for aggregated particle delivery is developed and an actuation scheme is proposed to deliver aggregated magnetic nanoparticles (MNPs) using a discontinuous asymmetrical magnetic actuation. The experimental results with a Y-shaped channel indicated the success of the proposed scheme in steering and disaggregation. The delivery performance of the developed scheme was examined using a realistic, three-dimensional (3D) vessel simulation. Furthermore, the proposed scheme enhanced the transport and uptake of MNPs across the BBB in mice. The scheme presented here facilitates the passage of particles across the BBB to the brain using an electromagnetic actuation scheme. PMID:29271927

A Novel Magnetic Actuation Scheme to Disaggregate Nanoparticles and Enhance Passage across the Blood-Brain Barrier.

PubMed

Hoshiar, Ali Kafash; Le, Tuan-Anh; Amin, Faiz Ul; Kim, Myeong Ok; Yoon, Jungwon

2017-12-22

The blood-brain barrier (BBB) hinders drug delivery to the brain. Despite various efforts to develop preprogramed actuation schemes for magnetic drug delivery, the unmodeled aggregation phenomenon limits drug delivery performance. This paper proposes a novel scheme with an aggregation model for a feed-forward magnetic actuation design. A simulation platform for aggregated particle delivery is developed and an actuation scheme is proposed to deliver aggregated magnetic nanoparticles (MNPs) using a discontinuous asymmetrical magnetic actuation. The experimental results with a Y-shaped channel indicated the success of the proposed scheme in steering and disaggregation. The delivery performance of the developed scheme was examined using a realistic, three-dimensional (3D) vessel simulation. Furthermore, the proposed scheme enhanced the transport and uptake of MNPs across the BBB in mice. The scheme presented here facilitates the passage of particles across the BBB to the brain using an electromagnetic actuation scheme.

Indirect three-dimensional printing: A method for fabricating polyurethane-urea based cardiac scaffolds.

PubMed

Hernández-Córdova, R; Mathew, D A; Balint, R; Carrillo-Escalante, H J; Cervantes-Uc, J M; Hidalgo-Bastida, L A; Hernández-Sánchez, F

2016-08-01

Biomaterial scaffolds are a key part of cardiac tissue engineering therapies. The group has recently synthesized a novel polycaprolactone based polyurethane-urea copolymer that showed improved mechanical properties compared with its previously published counterparts. The aim of this study was to explore whether indirect three-dimensional (3D) printing could provide a means to fabricate this novel, biodegradable polymer into a scaffold suitable for cardiac tissue engineering. Indirect 3D printing was carried out through printing water dissolvable poly(vinyl alcohol) porogens in three different sizes based on a wood-stack model, into which a polyurethane-urea solution was pressure injected. The porogens were removed, leading to soft polyurethane-urea scaffolds with regular tubular pores. The scaffolds were characterized for their compressive and tensile mechanical behavior; and their degradation was monitored for 12 months under simulated physiological conditions. Their compatibility with cardiac myocytes and performance in novel cardiac engineering-related techniques, such as aggregate seeding and bi-directional perfusion, was also assessed. The scaffolds were found to have mechanical properties similar to cardiac tissue, and good biocompatibility with cardiac myocytes. Furthermore, the incorporated cells preserved their phenotype with no signs of de-differentiation. The constructs worked well in perfusion experiments, showing enhanced seeding efficiency. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1912-1921, 2016. © 2016 Wiley Periodicals, Inc.

3D is not enough: Building up a cell instructive microenvironment for tumoral stroma microtissues.

PubMed

Brancato, Virginia; Garziano, Alessandro; Gioiella, Filomena; Urciuolo, Francesco; Imparato, Giorgia; Panzetta, Valeria; Fusco, Sabato; Netti, Paolo A

2017-01-01

We fabricated three-dimensional microtissues with the aim to replicate in vitro the composition and the functionalities of the tumor microenvironment. By arranging either normal fibroblasts (NF) or cancer-activated fibroblasts (CAF) in two different three dimensional (3D) configurations, two kinds of micromodules were produced: spheroids and microtissues. Spheroids were obtained by means of the traditional cell aggregation technique resulting in a 3D model characterized by high cell density and low amount of extracellular proteins. The microtissues were obtained by culturing cells into porous gelatin microscaffolds. In this latter configuration, cells assembled an intricate network of collagen, fibronectin and hyaluronic acid. We investigated the biophysical properties of both 3D models in terms of cell growth, metabolic activity, texture and composition of the extracellular matrix (via histological analysis and multiphoton imaging) and cell mechanical properties (via Particle Tracking Microrheology). In the spheroid models such biophysical properties remained unchanged regardless to the cell type used. In contrast, normal-microtissues and cancer-activated-microtissues displayed marked differences. CAF-microtissues possessed higher proliferation rate, superior contraction capability, different micro-rheological properties and an extracellular matrix richer in collagen fibronectin and hyaluronic acid. At last, multiphoton investigation revealed differences in the collagen network architecture. Taken together, these results suggested that despite to cell spheroids, microtissues better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the stromal element of the tumor tissues. This work concerns the engineering of tumor tissue in vitro. Tumor models serve as biological equivalent to study pathologic progression and to screen or validate the drugs efficacy. Tumor tissue is composed by malignant cells surviving in a microenvironment, or stroma. Stroma plays a pivotal role in cancer progression. Current in vitro models, i.e. spheroids, can't replicate the phenomena related to the tumor stroma remodeling. For this reason, to better replicate the tumor physiology in vitro that include functional and morphological changes, a novel 3D cancer model is proposed. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Three-dimensional control of Tetrahymena pyriformis using artificial magnetotaxis

NASA Astrophysics Data System (ADS)

Hyung Kim, Dal; Seung Soo Kim, Paul; Agung Julius, Anak; Jun Kim, Min

2012-01-01

We demonstrate three-dimensional control with the eukaryotic cell Tetrahymena pyriformis (T. pyriformis) using two sets of Helmholtz coils for xy-plane motion and a single electromagnet for z-direction motion. T. pyriformis is modified to have artificial magnetotaxis with internalized magnetite. To track the cell's z-axis position, intensity profiles of non-motile cells at varying distances from the focal plane are used. During vertical motion along the z-axis, the intensity difference is used to determine the position of the cell. The three-dimensional control of the live microorganism T. pyriformis as a cellular robot shows great potential for practical applications in microscale tasks, such as target transport and cell therapy.

Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

PubMed

Lavrov, Andrey I; Kosevich, Igor A

2016-02-01

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions. © 2016 Wiley Periodicals, Inc.

Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

PubMed

Wang, Xia; An, Qiang; Zhao, Bin; Guo, Jin Song; Huang, Yuan Sheng; Tian, Meng

2018-02-01

Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO 3 - -N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N 2 O and N 2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to β-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO 3 - -N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

Variations of floc morphology and extracellular organic matters (EOM) in relation to floc filterability under algae flocculation harvesting using polymeric titanium coagulants (PTCs).

PubMed

Zhang, Weijun; Song, Rongna; Cao, Bingdi; Yang, Xiaofang; Wang, Dongsheng; Fu, Xingmin; Song, Yao

2018-05-01

The work evaluated the algae cells removal efficiency using titanium salt coagulants with different degree of polymerization (PTCs), and the algae cells aggregates and extracellular organic matter (EOM) under chemical flocculation were investigated. The results indicated that PTCs performed well in algae cells flocculation and separation. The main mechanism using PTCs of low alkalisation degree for algae flocculation was associated with charge neutralization, while adsorption bridging and sweep flocculation was mainly responsible for algae removal by PTCs of high alkalisation degree treatment. In addition, the flocs formed by PTC 1.0 showed the best filtration property, and EOM reached the minimum at this time, indicating the flocs formed by PTC 1.0 were more compact than other PTCs, which can be confirmed by SEM analysis. Three-dimensional excitation emission matrix fluorescence (3D-EEM) and high performance size exclusion chromatography (HPSEC) revealed that the EOMs were removed under PTCs flocculation, which improved floc filterability. Copyright © 2018 Elsevier Ltd. All rights reserved.

Estimation of polydispersity in aggregating red blood cells by quantitative ultrasound backscatter analysis.

PubMed

de Monchy, Romain; Rouyer, Julien; Destrempes, François; Chayer, Boris; Cloutier, Guy; Franceschini, Emilie

2018-04-01

Quantitative ultrasound techniques based on the backscatter coefficient (BSC) have been commonly used to characterize red blood cell (RBC) aggregation. Specifically, a scattering model is fitted to measured BSC and estimated parameters can provide a meaningful description of the RBC aggregates' structure (i.e., aggregate size and compactness). In most cases, scattering models assumed monodisperse RBC aggregates. This study proposes the Effective Medium Theory combined with the polydisperse Structure Factor Model (EMTSFM) to incorporate the polydispersity of aggregate size. From the measured BSC, this model allows estimating three structural parameters: the mean radius of the aggregate size distribution, the width of the distribution, and the compactness of the aggregates. Two successive experiments were conducted: a first experiment on blood sheared in a Couette flow device coupled with an ultrasonic probe, and a second experiment, on the same blood sample, sheared in a plane-plane rheometer coupled to a light microscope. Results demonstrated that the polydisperse EMTSFM provided the best fit to the BSC data when compared to the classical monodisperse models for the higher levels of aggregation at hematocrits between 10% and 40%. Fitting the polydisperse model yielded aggregate size distributions that were consistent with direct light microscope observations at low hematocrits.

Validation of in vitro assays in three-dimensional human dermal constructs.

PubMed

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Shah, Siegfried; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

2018-05-01

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue ® , RealTime-Glo ™ MT, and CellTiter-Glo ® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

Myxococcus xanthus Developmental Cell Fate Production: Heterogeneous Accumulation of Developmental Regulatory Proteins and Reexamination of the Role of MazF in Developmental Lysis

PubMed Central

Lee, Bongsoo; Holkenbrink, Carina; Treuner-Lange, Anke

2012-01-01

Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazF's role in promoting PCD is an adaption to the mutant background strain used previously. PMID:22493014

Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution.

PubMed

Chiang, Ann-Shyn; Lin, Chih-Yung; Chuang, Chao-Chun; Chang, Hsiu-Ming; Hsieh, Chang-Huain; Yeh, Chang-Wei; Shih, Chi-Tin; Wu, Jian-Jheng; Wang, Guo-Tzau; Chen, Yung-Chang; Wu, Cheng-Chi; Chen, Guan-Yu; Ching, Yu-Tai; Lee, Ping-Chang; Lin, Chih-Yang; Lin, Hui-Hao; Wu, Chia-Chou; Hsu, Hao-Wei; Huang, Yun-Ann; Chen, Jing-Yi; Chiang, Hsin-Jung; Lu, Chun-Fang; Ni, Ru-Fen; Yeh, Chao-Yuan; Hwang, Jenn-Kang

2011-01-11

Animal behavior is governed by the activity of interconnected brain circuits. Comprehensive brain wiring maps are thus needed in order to formulate hypotheses about information flow and also to guide genetic manipulations aimed at understanding how genes and circuits orchestrate complex behaviors. To assemble this map, we deconstructed the adult Drosophila brain into approximately 16,000 single neurons and reconstructed them into a common standardized framework to produce a virtual fly brain. We have constructed a mesoscopic map and found that it consists of 41 local processing units (LPUs), six hubs, and 58 tracts covering the whole Drosophila brain. Despite individual local variation, the architecture of the Drosophila brain shows invariance for both the aggregation of local neurons (LNs) within specific LPUs and for the connectivity of projection neurons (PNs) between the same set of LPUs. An open-access image database, named FlyCircuit, has been constructed for online data archiving, mining, analysis, and three-dimensional visualization of all single neurons, brain-wide LPUs, their wiring diagrams, and neural tracts. We found that the Drosophila brain is assembled from families of multiple LPUs and their interconnections. This provides an essential first step in the analysis of information processing within and between neurons in a complete brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Summary of mathematical models for a conventional and vertical junction photoconverter

NASA Technical Reports Server (NTRS)

Heinbockel, J. H.

1986-01-01

The geometry and computer programming for mathematical models of a one-dimensional conventional photoconverter, a one-dimensional vertical junction photoconverter, a three-dimensional conventinal photoconverter, and a three-dimensional vertical junction solar cell are discussed.

In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

PubMed

Chen, X C; Liu, H; Li, H; Cheng, Y; Yang, L; Liu, Y F

2016-06-27

In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P < 0.01). By using a dynamic three-dimensional cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.

Effect of the LHCII pigment-protein complex aggregation on photovoltaic properties of sensitized TiO2 solar cells.

PubMed

Yang, Yiqun; Jankowiak, Ryszard; Lin, Chen; Pawlak, Krzysztof; Reus, Michael; Holzwarth, Alfred R; Li, Jun

2014-10-14

A modified dye-sensitized solar cell consisting of a thin TiO2 barrier layer sensitized with natural trimeric light-harvesting complex II (LHCII) from spinach was used as a biomimetic model to study the effects of LHCII aggregation on the photovoltaic properties. The aggregation of individual trimers induced molecular reorganization, which dramatically increased the photocurrent. The morphology of small- and large-size LHCII aggregates deposited on a surface was confirmed by atomic force microscopy. Enhanced LHCII immobilization was accomplished via electrostatic interaction with amine-functionalized photoanodes. The photocurrent responses of the assembled solar cells under illumination at three characteristic wavelength bands in the UV-Vis absorption spectra of LHCII solutions confirmed that a significant photocurrent was generated by LHCII photosensitizers. The enhanced photocurrent by large aggregated LHCII is shown to correlate with the quenching in the far-red fluorescence deriving from chlorophyll-chlorophyll charge transfer states that are effectively coupled with the TiO2 surface and thus inject electrons into the TiO2 conduction band. The large aggregated LHCII with more chlorophyll-chlorophyll charge transfer states is a much better sensitizer since it injects electrons more efficiently into the conduction band of TiO2 than the small aggregated LHCII mostly consisting of unquenched chlorophyll excited state. The assembled solar cells demonstrated remarkable stability in both aqueous buffer and acetonitrile electrolytes over 30 days.

Droplet-based microtumor model to assess cell-ECM interactions and drug resistance of gastric cancer cells.

PubMed

Jang, Minjeong; Koh, Ilkyoo; Lee, Seok Jae; Cheong, Jae-Ho; Kim, Pilnam

2017-01-27

Gastric cancer (GC) is a common aggressive malignant tumor with high incidence and mortality worldwide. GC is classified into intestinal and diffuse types according to the histo-morphological features. Because of distinctly different clinico-pathological features, new cancer therapy strategies and in vitro preclinical models for the two pathological variants of GC is necessary. Since extracellular matrix (ECM) influence the biological behavior of tumor cells, we hypothesized that GC might be more similarly modeled in 3D with matrix rather than in 2D. Herein, we developed a microfluidic-based a three-dimensional (3D) in vitro gastric cancer model, with subsequent drug resistance assay. AGS (intestinal type) and Hs746T (diffuse type) gastric cancer cell lines were encapsulated in collagen beads with high cellular viability. AGS exhibited an aggregation pattern with expansive growth, whereas Hs746T showed single-cell-level infiltration. Importantly, in microtumor models, epithelial-mesenchymal transition (EMT) and metastatic genes were upregulated, whereas E-cadherin was downregulated. Expression of ß-catenin was decreased in drug-resistant cells, and chemosensitivity toward the anticancer drug (5-FU) was observed in microtumors. These results suggest that in vitro microtumor models may represent a biologically relevant platform for studying gastric cancer cell biology and tumorigenesis, and for accelerating the development of novel therapeutic targets.

Development of a Single-Cell Migration and Extravasation Platform through Selective Surface Modification.

PubMed

Roberts, Steven A; Waziri, Allen E; Agrawal, Nitin

2016-03-01

Cell migration through three-dimensional (3D) tissue spaces is integral to many biological and pathological processes, including metastasis. Circulating tumor cells (CTCs) are phenotypically heterogeneous, and in vitro analysis of their extravasation behavior is often impeded by the inability to establish complex tissue-like extracellular matrix (ECM) environments and chemotactic gradients within microfluidic devices. We have developed a novel microfluidic strategy to manipulate surface properties of enclosed microchannels and create 3D ECM structures for real-time observation of individual migrating cells. The wettability of selective interconnected channels is controlled by a plasma pulse, enabling the incorporation of ECM exclusively within the transmigration regions. We applied this approach to collectively analyze CTC-endothelial adhesion, trans-endothelial migration, and subsequent motility of breast cancer cells (MDA-MB-231) through a 3D ECM under artificial gradients of SDF-1α. We observed migration velocities ranging from 5.12 to 12.8 μm/h, which closely correspond to single-cell migration in collagen blocks, but are significantly faster than the migration of cell aggregates. The compartmentalized microchannels separated by the porous ECM makes this in vitro assay versatile and suitable for a variety of applications such as inflammation studies, drug screening, and coculture interactions.

Dose-dependent photochemical/photothermal toxicity of indocyanine green-based therapy on three different cancer cell lines.

PubMed

Ruhi, Mustafa Kemal; Ak, Ayşe; Gülsoy, Murat

2018-03-01

The Food and Drug Administration-approved Indocyanine Green can be used as a photosensitizer to kill cancer cells selectively. Although indocyanine green is advantageous as a photosensitizer in terms of strong absorption in the near-infrared region, indocyanine green-based cancer treatment is still not approved as a clinical method. Some reasons for this are aggregation at high concentrations, rapid clearance of the photosensitizer from the body, low singlet oxygen quantum yield, and the uncertainty concerning its action mechanism. This in vitro study focuses on two of these points: "what is the cell inhibition mechanism of indocyanine green-based therapy?" and "how the dose-dependent aggregation problem of indocyanine green alters its cell inhibition efficiency?" The following experiments were conducted to provide insight into these points. Nontoxic doses of indocyanine green and near-infrared laser were determined. The aggregation behavior of indocyanine green was verified through experiments. The singlet oxygen quantum yield of indocyanine green at different concentrations were calculated. Various indocyanine green and energy densities of near-infrared light were applied to prostate cancer, neuroblastoma, and colon cancer cells. An MTT assay was performed at the end of the first, second, and third days following the treatments to determine the cell viability. Temperature changes in the medium during laser exposure were recorded. ROS generation following the treatment was verified by using a Total Reactive Oxygen Species detection kit. An apoptosis detection test was performed to establish the cell death mechanism and, finally, the cellular uptakes of the three different cells were measured. According to the results, indocyanine green-based therapy causes cell viability decrease for three cancer cell lines by means of excessive reactive oxygen species production. Different cells have different sensitivities to the therapy possibly because of the differentiation level and structural differences. The singlet oxygen generation of indocyanine green decreases at high concentrations because of aggregation. Nevertheless, better cancer cell killing effect was observed at higher photosensitizer concentrations. This result reveals that the cellular uptake of indocyanine green was determinant for better cancer cell inhibition. Copyright © 2018 Elsevier B.V. All rights reserved.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

PubMed Central

Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

2016-01-01

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

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

Kim, G. H.; Pesaran, A.; Spotnitz, R.

To understand further the thermal abuse behavior of large format Li-ion batteries for automotive applications, the one-dimensional modeling approach formulated by Hatchard et al. was reproduced. Then it was extended to three dimensions so we could consider the geometrical features, which are critical in large cells for automotive applications. The three-dimensional model captures the shapes and dimensions of cell components and the spatial distributions of materials and temperatures, and is used to simulate oven tests, and to determine how a local hot spot can propagate through the cell. In simulations of oven abuse testing of cells with cobalt oxide cathodemore » and graphite anode with standard LiPF6 electrolyte, the three-dimensional model predicts that thermal runaway will occur sooner or later than the lumped model, depending on the size of the cell. The model results showed that smaller cells reject heat faster than larger cells; this may prevent them from going into thermal runaway under identical abuse conditions. In simulations of local hot spots inside a large cylindrical cell, the three-dimensional model predicts that the reactions initially propagate in the azimuthal and longitudinal directions to form a hollow cylinder-shaped reaction zone.« less

Chemical colloids versus biological colloids: a comparative study for the elucidation of the mechanism of protein fiber formation

NASA Technical Reports Server (NTRS)

Xu, Shaohua; Wu, David; Arnsdorf, Morton; Johnson, Robert; Getz, Godfrey S.; Cabana, Veneracion G.

2005-01-01

Fiber formation from murine serum amyloid A1 (SAA) was compared to the linear aggregation and fiber formation of colloidal gold particles. Here we report the similarities of these processes. Upon incubation with acetic acid, SAA misfolds and adopts a new conformation, which we termed saa. saa apparently is less soluble than SAA in aqueous solution; it aggregates and forms nucleation units and then fibers. The fibers appear as a string of the nucleation units. Additionally, an external electric field promotes saa fiber formation. These properties of saa are reminiscent of colloidal gold formation from gold ions and one-dimensional aggregation of the gold colloids. Colloidal gold particles were also found to be capable of aggregating one-dimensionally under an electric field or in the presence of polylysine. These gold fibers resembled in structure that of saa fibers. In summary, protein aggregation and formation of fibers appear to follow the generalized principles derived in colloidal science for the aggregation of atoms and molecules, including polymers such as polypeptides. The analysis of colloidal gold formation and of one-dimensional aggregation provides a simple model system for the elucidation of some aspects of protein fiber formation.

A light-trapping strategy for nanocrystalline silicon thin-film solar cells using three-dimensionally assembled nanoparticle structures.

PubMed

Ha, Kyungyeon; Jang, Eunseok; Jang, Segeun; Lee, Jong-Kwon; Jang, Min Seok; Choi, Hoseop; Cho, Jun-Sik; Choi, Mansoo

2016-02-05

We report three-dimensionally assembled nanoparticle structures inducing multiple plasmon resonances for broadband light harvesting in nanocrystalline silicon (nc-Si:H) thin-film solar cells. A three-dimensional multiscale (3DM) assembly of nanoparticles generated using a multi-pin spark discharge method has been accomplished over a large area under atmospheric conditions via ion-assisted aerosol lithography. The multiscale features of the sophisticated 3DM structures exhibit surface plasmon resonances at multiple frequencies, which increase light scattering and absorption efficiency over a wide spectral range from 350-1100 nm. The multiple plasmon resonances, together with the antireflection functionality arising from the conformally deposited top surface of the 3D solar cell, lead to a 22% and an 11% improvement in power conversion efficiency of the nc-Si:H thin-film solar cells compared to flat cells and cells employing nanoparticle clusters, respectively. Finite-difference time-domain simulations were also carried out to confirm that the improved device performance mainly originates from the multiple plasmon resonances generated from three-dimensionally assembled nanoparticle structures.

Characterisation of cell-substrate interactions between Schwann cells and three-dimensional fibrin hydrogels containing orientated nanofibre topographical cues.

PubMed

Hodde, Dorothee; Gerardo-Nava, José; Wöhlk, Vanessa; Weinandy, Stefan; Jockenhövel, Stefan; Kriebel, Andreas; Altinova, Haktan; Steinbusch, Harry W M; Möller, Martin; Weis, Joachim; Mey, Jörg; Brook, Gary A

2016-02-01

The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Titanate and titania nanostructures and nanostructure assemblies, and methods of making same

DOEpatents

Wong, Stanislaus S.; Mao, Yuanbing

2016-06-14

The invention relates to nanomaterial's and assemblies including, a micrometer-scale spherical aggregate comprising: a plurality of one-dimensional nanostructures comprising titanium and oxygen, wherein the one-dimensional nanostructures radiate from a hollow central core thereby forming a spherical aggregate.

Titanate and titania nanostructures and nanostructure assemblies, and methods of making same

DOEpatents

Wong, Stanislaus S; Mao, Yuanbing

2013-05-14

The invention relates to nanomaterials and assemblies including, a micrometer-scale spherical aggregate comprising: a plurality of one-dimensional nanostructures comprising titanium and oxygen, wherein the one-dimensional nanostructures radiate from a hollow central core thereby forming a spherical aggregate.

Three-Dimensional Simulation of Ultrasound-Induced Microalgal Cell Disruption.

PubMed

Wang, M; Yuan, W; Hale, Andy

2016-03-01

The three-dimensional distribution (x, y, and z) of ultrasound-induced microalgal cell disruption in a sonochemical reactor was predicted by solving the Helmholtz equation using a three-dimensional acoustic module in the COMSOL Multiphysics software. The simulated local ultrasound pressure at any given location (x, y, and z) was found to correlate with cell disruption of a freshwater alga, Scenedesmus dimorphus, represented by the change of algal cell particle/debris concentration, chlorophyll-a fluorescence density (CAFD), and Nile red stained lipid fluorescence density (LFD), which was also validated by the model reaction of potassium iodide oxidation (the Weissler reaction). Furthermore, the effect of ultrasound power intensity and processing duration on algal cell disruption was examined to address the limitation of the model.

Anisotropic encoding of three-dimensional space by place cells and grid cells

PubMed Central

Hayman, R.; Verriotis, M.; Jovalekic, A.; Fenton, A.A.; Jeffery, K.J.

2011-01-01

The subjective sense of space may result in part from the combined activity of place cells, in the hippocampus, and grid cells in posterior cortical regions such as entorhinal cortex and pre/parasubiculum. In horizontal planar environments, place cells provide focal positional information while grid cells supply odometric (distance-measuring) information. How these cells operate in three dimensions is unknown, even though the real world is three–dimensional. The present study explored this issue in rats exploring two different kinds of apparatus, a climbing wall (the “pegboard”) and a helix. Place and grid cell firing fields had normal horizontal characteristics but were elongated vertically, with grid fields forming stripes. It appears that grid cell odometry (and by implication path integration) is impaired/absent in the vertical domain, at least when the animal itself remains horizontal. These findings suggest that the mammalian encoding of three-dimensional space is anisotropic. PMID:21822271

Computer simulation of plasma and N-body problems

NASA Technical Reports Server (NTRS)

Harries, W. L.; Miller, J. B.

1975-01-01

The following FORTRAN language computer codes are presented: (1) efficient two- and three-dimensional central force potential solvers; (2) a three-dimensional simulator of an isolated galaxy which incorporates the potential solver; (3) a two-dimensional particle-in-cell simulator of the Jeans instability in an infinite self-gravitating compressible gas; and (4) a two-dimensional particle-in-cell simulator of a rotating self-gravitating compressible gaseous system of which rectangular coordinate and superior polar coordinate versions were written.

Low temperature exciton dynamics and structural changes in perylene bisimide aggregates

NASA Astrophysics Data System (ADS)

Wolter, Steffen; Magnus Westphal, Karl; Hempel, Magdalena; Würthner, Frank; Kühn, Oliver; Lochbrunner, Stefan

2017-09-01

The temperature dependent exciton dynamics of J-aggregates formed by a perylene bisimide dye is investigated down to liquid nitrogen temperature (77 K) by femtosecond pump-probe spectroscopy. The analysis of the transient absorption data using a diffusion model for the excitons does not only reveal an overall decrease of the exciton mobility, but also a change in the dimensionality of the exciton transport at low temperatures. This change in dimensionality is further investigated by kinetic Monte Carlo simulations, identifying weakly interlinked one-dimensional aggregate chains as the most likely structure at low temperatures. This causes the exciton transport to be highly anisotropic.

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns.

PubMed

Barrila, Jennifer; Yang, Jiseon; Crabbé, Aurélie; Sarker, Shameema F; Liu, Yulong; Ott, C Mark; Nelman-Gonzalez, Mayra A; Clemett, Simon J; Nydam, Seth D; Forsyth, Rebecca J; Davis, Richard R; Crucian, Brian E; Quiriarte, Heather; Roland, Kenneth L; Brenneman, Karen; Sams, Clarence; Loscher, Christine; Nickerson, Cheryl A

2017-01-01

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella , we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Real-time imaging of Huntingtin aggregates diverting target search and gene transcription

PubMed Central

Li, Li; Liu, Hui; Dong, Peng; Li, Dong; Legant, Wesley R; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert; Liu, Zhe

2016-01-01

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001 PMID:27484239

Ray tracing a three dimensional scene using a grid

DOEpatents

Wald, Ingo; Ize, Santiago; Parker, Steven G; Knoll, Aaron

2013-02-26

Ray tracing a three-dimensional scene using a grid. One example embodiment is a method for ray tracing a three-dimensional scene using a grid. In this example method, the three-dimensional scene is made up of objects that are spatially partitioned into a plurality of cells that make up the grid. The method includes a first act of computing a bounding frustum of a packet of rays, and a second act of traversing the grid slice by slice along a major traversal axis. Each slice traversal includes a first act of determining one or more cells in the slice that are overlapped by the frustum and a second act of testing the rays in the packet for intersection with any objects at least partially bounded by the one or more cells overlapped by the frustum.

Strategic planning for aircraft noise route impact analysis: A three dimensional approach

NASA Technical Reports Server (NTRS)

Bragdon, C. R.; Rowan, M. J.; Ahuja, K. K.

1993-01-01

The strategic routing of aircraft through navigable and controlled airspace to minimize adverse noise impact over sensitive areas is critical in the proper management and planning of the U.S. based airport system. A major objective of this phase of research is to identify, inventory, characterize, and analyze the various environmental, land planning, and regulatory data bases, along with potential three dimensional software and hardware systems that can be potentially applied for an impact assessment of any existing or planned air route. There are eight data bases that have to be assembled and developed in order to develop three dimensional aircraft route impact methodology. These data bases which cover geographical information systems, sound metrics, land use, airspace operational control measures, federal regulations and advisories, census data, and environmental attributes have been examined and aggregated. A three dimensional format is necessary for planning, analyzing space and possible noise impact, and formulating potential resolutions. The need to develop this three dimensional approach is essential due to the finite capacity of airspace for managing and planning a route system, including airport facilities. It appears that these data bases can be integrated effectively into a strategic aircraft noise routing system which should be developed as soon as possible, as part of a proactive plan applied to our FAA controlled navigable airspace for the United States.

Geotechnical Characteristics and Stability Analysis of Rock-Soil Aggregate Slope at the Gushui Hydropower Station, Southwest China

PubMed Central

Shi, Chong; Xu, Fu-gang

2013-01-01

Two important features of the high slopes at Gushui Hydropower Station are layered accumulations (rock-soil aggregate) and multilevel toppling failures of plate rock masses; the Gendakan slope is selected for case study in this paper. Geological processes of the layered accumulation of rock and soil particles are carried out by the movement of water flow; the main reasons for the toppling failure of plate rock masses are the increasing weight of the upper rock-soil aggregate and mountain erosion by river water. Indoor triaxial compression test results show that, the cohesion and friction angle of the rock-soil aggregate decreased with the increasing water content; the cohesion and the friction angle for natural rock-soil aggregate are 57.7 kPa and 31.3° and 26.1 kPa and 29.1° for saturated rock-soil aggregate, respectively. The deformation and failure mechanism of the rock-soil aggregate slope is a progressive process, and local landslides will occur step by step. Three-dimensional limit equilibrium analysis results show that the minimum safety factor of Gendakan slope is 0.953 when the rock-soil aggregate is saturated, and small scale of landslide will happen at the lower slope. PMID:24082854

Geotechnical characteristics and stability analysis of rock-soil aggregate slope at the Gushui Hydropower Station, southwest China.

PubMed

Zhou, Jia-wen; Shi, Chong; Xu, Fu-gang

2013-01-01

Two important features of the high slopes at Gushui Hydropower Station are layered accumulations (rock-soil aggregate) and multilevel toppling failures of plate rock masses; the Gendakan slope is selected for case study in this paper. Geological processes of the layered accumulation of rock and soil particles are carried out by the movement of water flow; the main reasons for the toppling failure of plate rock masses are the increasing weight of the upper rock-soil aggregate and mountain erosion by river water. Indoor triaxial compression test results show that, the cohesion and friction angle of the rock-soil aggregate decreased with the increasing water content; the cohesion and the friction angle for natural rock-soil aggregate are 57.7 kPa and 31.3° and 26.1 kPa and 29.1° for saturated rock-soil aggregate, respectively. The deformation and failure mechanism of the rock-soil aggregate slope is a progressive process, and local landslides will occur step by step. Three-dimensional limit equilibrium analysis results show that the minimum safety factor of Gendakan slope is 0.953 when the rock-soil aggregate is saturated, and small scale of landslide will happen at the lower slope.

Workshop on Alternatives to Animals in Research Held in Suffield, Ralston, Alberta on 16-17 September 1987

DTIC Science & Technology

1989-10-01

UNCLASSIFIED UNCLASSIFIED 48 SUMMARY OF OP AND CARBAMATE INTERACTIONS IN THE ACUTE INHIBITION OF CHOLINESTERASE ACTIVITY IN RAT BRAIN NEURAL CELL AGGREGATE...for the past three years, we have been study- ing the toxicity of a number of organophosphates and carbamates on neural cell aggregate cultures. The...the temporary acute exposure to high concentrations of the chemical. In the occupational setting, accidental discharge may occur, resulting in acute

Microfluidic engineering of neural stem cell niches for fate determination

PubMed Central

Ma, Jingyun; Li, Na; Wang, Liang; Shen, Liming; Sun, Yu; Wang, Yajun; Zhao, Jingyuan; Wei, Wenjuan; Ren, Yan; Liu, Jing

2017-01-01

Neural stem cell (NSC) transplantation has great therapeutic potential for neurodegenerative diseases and central nervous system injuries. Successful NSC replacement therapy requires precise control over the cellular behaviors. However, the regulation of NSC fate is largely unclear, which severely restricts the potential clinical applications. To develop an effective model, we designed an assembled microfluidic system to engineer NSC niches and assessed the effects of various culture conditions on NSC fate determination. Five types of NSC microenvironments, including two-dimensional (2D) cellular monolayer culture, 2D cellular monolayer culture on the extracellular matrix (ECM), dispersed cells in the ECM, three-dimensional (3D) spheroid aggregates, and 3D spheroids cultured in the ECM, were constructed within an integrated microfluidic chip simultaneously. In addition, we evaluated the influence of static and perfusion culture on NSCs. The efficiency of this approach was evaluated comprehensively by characterization of NSC viability, self-renewal, proliferation, and differentiation into neurons, astrocytes, or oligodendrocytes. Differences in the status and fate of NSCs governed by the culture modes and micro-niches were analyzed. NSCs in the microfluidic device demonstrated good viability, the 3D culture in the ECM facilitated NSC self-renewal and proliferation, and 2D culture in the static state and spheroid culture under perfusion conditions benefited NSC differentiation. Regulation of NSC self-renewal and differentiation on this microfluidic device could provide NSC-based medicinal products and references for distinct nerve disease therapy. PMID:28798841

Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

PubMed

Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

2014-09-01

This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A hybrid nanostructure of platinum-nanoparticles/graphitic-nanofibers as a three-dimensional counter electrode in dye-sensitized solar cells.

PubMed

Hsieh, Chien-Kuo; Tsai, Ming-Chi; Su, Ching-Yuan; Wei, Sung-Yen; Yen, Ming-Yu; Ma, Chen-Chi M; Chen, Fu-Rong; Tsai, Chuen-Horng

2011-11-07

We directly synthesized a platinum-nanoparticles/graphitic-nanofibers (PtNPs/GNFs) hybrid nanostructure on FTO glass. We applied this structure as a three-dimensional counter electrode in dye-sensitized solar cells (DSSCs), and investigated the cells' photoconversion performance. This journal is © The Royal Society of Chemistry 2011

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model.

PubMed

Zhang, Wenli; Li, Caibin; Baguley, Bruce C; Zhou, Fang; Zhou, Weisai; Shaw, John P; Wang, Zhen; Wu, Zimei; Liu, Jianping

2016-12-15

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy. Copyright © 2016 Elsevier Inc. All rights reserved.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydro-focusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-Focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feedback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was micro-fabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, micro-fabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily micro-fabricated and integrated with other polymer microfluidic structures.

Physical Mechanisms Driving Cell Sorting in Hydra.

PubMed

Cochet-Escartin, Olivier; Locke, Tiffany T; Shi, Winnie H; Steele, Robert E; Collins, Eva-Maria S

2017-12-19

Cell sorting, whereby a heterogeneous cell mixture organizes into distinct tissues, is a fundamental patterning process in development. Hydra is a powerful model system for carrying out studies of cell sorting in three dimensions, because of its unique ability to regenerate after complete dissociation into individual cells. The physicists Alfred Gierer and Hans Meinhardt recognized Hydra's self-organizing properties more than 40 years ago. However, what drives cell sorting during regeneration of Hydra from cell aggregates is still debated. Differential motility and differential adhesion have been proposed as driving mechanisms, but the available experimental data are insufficient to distinguish between these two. Here, we answer this longstanding question by using transgenic Hydra expressing fluorescent proteins and a multiscale experimental and numerical approach. By quantifying the kinematics of single cell and whole aggregate behaviors, we show that no differences in cell motility exist among cell types and that sorting dynamics follow a power law with an exponent of ∼0.5. Additionally, we measure the physical properties of separated tissues and quantify their viscosities and surface tensions. Based on our experimental results and numerical simulations, we conclude that tissue interfacial tensions are sufficient to explain cell sorting in aggregates of Hydra cells. Furthermore, we demonstrate that the aggregate's geometry during sorting is key to understanding the sorting dynamics and explains the exponent of the power law behavior. Our results answer the long standing question of the physical mechanisms driving cell sorting in Hydra cell aggregates. In addition, they demonstrate how powerful this organism is for biophysical studies of self-organization and pattern formation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Lateral assembly of oxidized graphene flakes into large-scale transparent conductive thin films with a three-dimensional surfactant 4-sulfocalix[4]arene

PubMed Central

Sundramoorthy, Ashok K.; Wang, Yilei; Wang, Jing; Che, Jianfei; Thong, Ya Xuan; Lu, Albert Chee W.; Chan-Park, Mary B.

2015-01-01

Graphene is a promising candidate material for transparent conductive films because of its excellent conductivity and one-carbon-atom thickness. Graphene oxide flakes prepared by Hummers method are typically several microns in size and must be pieced together in order to create macroscopic films. We report a macro-scale thin film fabrication method which employs a three-dimensional (3-D) surfactant, 4-sulfocalix[4]arene (SCX), as a lateral aggregating agent. After electrochemical exfoliation, the partially oxidized graphene (oGr) flakes are dispersed with SCX. The SCX forms micelles, which adsorb on the oGr flakes to enhance their dispersion, also promote aggregation into large-scale thin films under vacuum filtration. A thin oGr/SCX film can be shaved off from the aggregated oGr/SCX cake by immersing the cake in water. The oGr/SCX thin-film floating on the water can be subsequently lifted from the water surface with a substrate. The reduced oGr (red-oGr) films can be as thin as 10−20 nm with a transparency of >90% and sheet resistance of 890 ± 47 kΩ/sq. This method of electrochemical exfoliation followed by SCX-assisted suspension and hydrazine reduction, avoids using large amounts of strong acid (unlike Hummers method), is relatively simple and can easily form a large scale conductive and transparent film from oGr/SCX suspension. PMID:26040436

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting^.

PubMed

Moldovan, Nicanor I; Hibino, Narutoshi; Nakayama, Koichi

2017-06-01

Bioprinting is a technology with the prospect to change the way many diseases are treated, by replacing the damaged tissues with live de novo created biosimilar constructs. However, after more than a decade of incubation and many proofs of concept, the field is still in its infancy. The current stagnation is the consequence of its early success: the first bioprinters, and most of those that followed, were modified versions of the three-dimensional printers used in additive manufacturing, redesigned for layer-by-layer dispersion of biomaterials. In all variants (inkjet, microextrusion, or laser assisted), this approach is material ("scaffold") dependent and energy intensive, making it hardly compatible with some of the intended biological applications. Instead, the future of bioprinting may benefit from the use of gentler scaffold-free bioassembling methods. A substantial body of evidence has accumulated, indicating this is possible by use of preformed cell spheroids, which have been assembled in cartilage, bone, and cardiac muscle-like constructs. However, a commercial instrument capable to directly and precisely "print" spheroids has not been available until the invention of the microneedles-based ("Kenzan") spheroid assembling and the launching in Japan of a bioprinter based on this method. This robotic platform laces spheroids into predesigned contiguous structures with micron-level precision, using stainless steel microneedles ("kenzans") as temporary support. These constructs are further cultivated until the spheroids fuse into cellular aggregates and synthesize their own extracellular matrix, thus attaining the needed structural organization and robustness. This novel technology opens wide opportunities for bioengineering of tissues and organs.

Three-dimensional in vitro cancer spheroid models for Photodynamic Therapy: Strengths and Opportunities

NASA Astrophysics Data System (ADS)

Evans, Conor

2015-03-01

Three dimensional, in vitro spheroid cultures offer considerable utility for the development and testing of anticancer photodynamic therapy regimens. More complex than monolayer cultures, three-dimensional spheroid systems replicate many of the important cell-cell and cell-matrix interactions that modulate treatment response in vivo. Simple enough to be grown by the thousands and small enough to be optically interrogated, spheroid cultures lend themselves to high-content and high-throughput imaging approaches. These advantages have enabled studies investigating photosensitizer uptake, spatiotemporal patterns of therapeutic response, alterations in oxygen diffusion and consumption during therapy, and the exploration of mechanisms that underlie therapeutic synergy. The use of quantitative imaging methods, in particular, has accelerated the pace of three-dimensional in vitro photodynamic therapy studies, enabling the rapid compilation of multiple treatment response parameters in a single experiment. Improvements in model cultures, the creation of new molecular probes of cell state and function, and innovations in imaging toolkits will be important for the advancement of spheroid culture systems for future photodynamic therapy studies.

Identification of the gene for disaggregatase from Methanosarcina mazei.

PubMed

Osumi, Naoki; Kakehashi, Yoshihiro; Matsumoto, Shiho; Nagaoka, Kazunari; Sakai, Junichi; Miyashita, Kiyotaka; Kimura, Makoto; Asakawa, Susumu

2008-12-01

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates of Methanosarcina mazei to single cells, were determined for three strains of M. mazei (S-6(T), LYC and TMA). The dag genes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genus Methanosarcina and may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed in Escherichia coli and preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed that dag was transcribed to mRNA in M. mazei LYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change of Methanosarcina spp. from aggregate to single cells has been identified.

Quantitative evaluation of morphological changes in activated platelets in vitro using digital holographic microscopy.

PubMed

Kitamura, Yutaka; Isobe, Kazushige; Kawabata, Hideo; Tsujino, Tetsuhiro; Watanabe, Taisuke; Nakamura, Masayuki; Toyoda, Toshihisa; Okudera, Hajime; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2018-06-18

Platelet activation and aggregation have been conventionally evaluated using an aggregometer. However, this method is suitable for short-term but not long-term quantitative evaluation of platelet aggregation, morphological changes, and/or adhesion to specific materials. The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction. Thus, we aim to validate its applicability in quantitatively evaluating changes in cell morphology, especially in the aggregation and spreading of activated platelets, thus modifying typical image analysis procedures to suit aggregated platelets. Freshly prepared platelet-rich plasma was washed with phosphate-buffered saline and treated with 0.1% CaCl 2 . Platelets were then fixed and subjected to DHM, scanning electron microscopy (SEM), atomic force microscopy, optical microscopy, and flow cytometry (FCM). Tightly aggregated platelets were identified as single cells. Data obtained from time-course experiments were plotted two-dimensionally according to the average optical thickness versus attachment area and divided into four regions. The majority of the control platelets, which supposedly contained small and round platelets, were distributed in the lower left region. As activation time increased, however, this population dispersed toward the upper right region. The distribution shift demonstrated by DHM was essentially consistent with data obtained from SEM and FCM. Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth

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

Luef, Birgit; Fakra, Sirine C.; Csencsits, Roseann

2013-02-04

Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III) bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Further, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on sitemore » and subsequently examined using correlated 2- and 3- dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). Most cells had their outer membranes decorated with up to 150 nm diameter aggregates composed of a few nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well studied group of FeRB. STXM results at the Fe L2,3 absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)-Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell-surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.« less

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

PubMed

Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio

2014-01-01

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Pathogen propagation in cultured three-dimensional tissue mass

NASA Technical Reports Server (NTRS)

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

2000-01-01

A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.

Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots.

PubMed

Hirata, Yoshito; Oda, Arisa; Ohta, Kunihiro; Aihara, Kazuyuki

2016-10-11

Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.

Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots

NASA Astrophysics Data System (ADS)

Hirata, Yoshito; Oda, Arisa; Ohta, Kunihiro; Aihara, Kazuyuki

2016-10-01

Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.

Applications of molecular self-assembly in tissue engineering

NASA Astrophysics Data System (ADS)

Harrington, Daniel Anton

This thesis studied the application of three self-assembling molecular systems, as potential biomaterials for tissue engineering applications. Cholesteryl-(L-lactic acid)n molecules form thermotropic liquid crystals, which could be coated onto the inner and outer pores of biodegradable PLLA scaffolds, while retaining the lamellar order of the neat material. Primary bovine chondrocytes were cultured on these structures, demonstrating improved attachment and extended retention of phenotype on the C-LA-coated scaffolds. No difference in fibronectin adsorption to C-LA and PLLA surfaces was observed, suggesting a strong role for cholesterol in influencing cell phenotype. A family of peptide-amphiphiles, bearing the "RGD" adhesion sequence from fibronectin, was also assessed in the contexts of cartilage and bladder repair. These molecules self-assemble into one-dimensional fibers, with diameters of 6--8 nm, and lengths of 500 nm or greater. Chondrocytes were seeded and cultured on covalently-crosslinked PA gels and embedded within calcium-triggered PA gels. Cells became dormant over time, but remained viable, suggesting an inappropriate display of the adhesion sequence to cells. A family of "branched" PA molecules with lysine dendron headgroups was designed, in an effort to increase the spatial separation between molecules in the assembled state, and to theoretically improve epitope accessibility. These molecules coated reliably onto PGA fiber scaffolds, and dramatically increased the attachment of human bladder smooth muscle cells, possibly through better epitope display or electrostatic attraction. They also formed strong gels with several negatively-charged biologically-relevant macromolecules. In a third system, amphiphilic segmented dendrimers based on phenylene vinylene and L-lysine entered cells through an endocytic pathway with no discernible toxic effect on cell proliferation or morphology. These amphiphiles formed complex aggregates in aqueous solution, likely an equilibrium state of micelles (5--10 nm) and vesicles (25--35 nm). A pyrene analogue was shown to lyse cells, which correlated with the molecule's reduced propensity to form strong aggregates in aqueous solution. Other amino acid segments were substituted for L-lysine, and only those amphiphiles with basic residues were efficiently taken in by cells. For all three self-assembling systems, their nanoscale organization and their interaction with biological systems were directly related to the chemical nature of their constituent building blocks.

Three-dimensional graphene foam as a biocompatible and conductive scaffold for neural stem cells

PubMed Central

Li, Ning; Zhang, Qi; Gao, Song; Song, Qin; Huang, Rong; Wang, Long; Liu, Liwei; Dai, Jianwu; Tang, Mingliang; Cheng, Guosheng

2013-01-01

Neural stem cell (NSC) based therapy provides a promising approach for neural regeneration. For the success of NSC clinical application, a scaffold is required to provide three-dimensional (3D) cell growth microenvironments and appropriate synergistic cell guidance cues. Here, we report the first utilization of graphene foam, a 3D porous structure, as a novel scaffold for NSCs in vitro. It was found that three-dimensional graphene foams (3D-GFs) can not only support NSC growth, but also keep cell at an active proliferation state with upregulation of Ki67 expression than that of two-dimensional graphene films. Meanwhile, phenotypic analysis indicated that 3D-GFs can enhance the NSC differentiation towards astrocytes and especially neurons. Furthermore, a good electrical coupling of 3D-GFs with differentiated NSCs for efficient electrical stimulation was observed. Our findings implicate 3D-GFs could offer a powerful platform for NSC research, neural tissue engineering and neural prostheses. PMID:23549373

Osteoinduction and proliferation of bone-marrow stromal cells in three-dimensional poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds.

PubMed

Wang, Ting; Yang, Xiaoyan; Qi, Xin; Jiang, Chaoyin

2015-05-08

Osteoinduction and proliferation of bone-marrow stromal cells (BMSCs) in three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds have not been studied throughly and are technically challenging. This study aimed to optimize nanocomposites of 3D PCL scaffolds to provide superior adhesion, proliferation and differentiation environment for BMSCs in this scenario. BMSCs were isolated and cultured in a novel 3D tissue culture poly(ε-caprolactone) (PCL) scaffold coated with poly-lysine, hydroxyapatite (HAp), collagen and HAp/collagen. Cell morphology was observed and BMSC biomarkers for osteogenesis, osteoblast differentiation and activation were analyzed. Scanning Electron Microscope (SEM) micrographs showed that coating materials were uniformly deposited on the surface of PCL scaffolds and BMSCs grew and aggregated to form clusters during 3D culture. Both mRNA and protein levels of the key players of osteogenesis and osteoblast differentiation and activation, including runt-related transcription factor 2 (Runx2), alkaline phosphates (ALP), osterix, osteocalcin, and RANKL, were significantly higher in BMSCs seeded in PCL scaffolds coated with HAp or HAp/collagen than those seeded in uncoated PCL scaffolds, whereas the expression levels were not significantly different in collagen or poly-lysine coated PCL scaffolds. In addition, poly-lysine, collagen, HAp/collagen, and HAp coated PCL scaffolds had significantly more viable cells than uncoated PCL scaffolds, especially scaffolds with HAp/collagen and collagen-alone coatings. That BMSCs in HAp or HAp/collagen PCL scaffolds had remarkably higher ALP activities than those in collagen-coated alone or uncoated PCL scaffolds indicating higher osteogenic differentiation levels of BMSCs in HAp or HAp/collagen PCL scaffolds. Moreover, morphological changes of BMSCs after four-week of 3D culture confirmed that BMSCs successfully differentiated into osteoblast with spread-out phenotype in HAp/collagen coated PCL scaffolds. This study showed a proof of concept for preparing biomimetic 3D poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds with excellent osteoinduction and proliferation capacity for bone regeneration.

Two-dimensional numerical modeling for separation of deformable cells using dielectrophoresis.

PubMed

Ye, Ting; Li, Hua; Lam, K Y

2015-02-01

In this paper, we numerically explore the possibility of separating two groups of deformable cells, by a very small dielectrophoretic (DEP) microchip with the characteristic length of several cell diameters. A 2D two-fluid model is developed to describe the separation process, where three types of forces are considered, the aggregation force for cell-cell interaction, the deformation force for cell deformation, and the DEP force for cell dielectrophoresis. As a model validation, we calculate the levitation height of a cell subject to DEP force, and compare it with the experimental data. After that, we simulate the separation of two groups of cells with different dielectric properties at high and low frequencies, respectively. The simulation results show that the deformable cells can be separated successfully by a very small DEP microchip, according to not only their different permittivities at the high frequency, but also their different conductivities at the low frequency. In addition, both two groups of cells have a shape deformation from an original shape to a lopsided slipper shape during the separation process. It is found that the cell motion is mainly determined by the DEP force arising from the electric field, causing the cells to deviate from the centerline of microchannel. However, the cell deformation is mainly determined by the deformation force arising from the fluid flow, causing the deviated cells to undergo an asymmetric motion with the deformation of slipper shape. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of streams in myxobacteria aggregate formation

NASA Astrophysics Data System (ADS)

Kiskowski, Maria A.; Jiang, Yi; Alber, Mark S.

2004-10-01

Cell contact, movement and directionality are important factors in biological development (morphogenesis), and myxobacteria are a model system for studying cell-cell interaction and cell organization preceding differentiation. When starved, thousands of myxobacteria cells align, stream and form aggregates which later develop into round, non-motile spores. Canonically, cell aggregation has been attributed to attractive chemotaxis, a long range interaction, but there is growing evidence that myxobacteria organization depends on contact-mediated cell-cell communication. We present a discrete stochastic model based on contact-mediated signaling that suggests an explanation for the initialization of early aggregates, aggregation dynamics and final aggregate distribution. Our model qualitatively reproduces the unique structures of myxobacteria aggregates and detailed stages which occur during myxobacteria aggregation: first, aggregates initialize in random positions and cells join aggregates by random walk; second, cells redistribute by moving within transient streams connecting aggregates. Streams play a critical role in final aggregate size distribution by redistributing cells among fewer, larger aggregates. The mechanism by which streams redistribute cells depends on aggregate sizes and is enhanced by noise. Our model predicts that with increased internal noise, more streams would form and streams would last longer. Simulation results suggest a series of new experiments.

Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering.

PubMed

Jabbarzadeh, Ehsan; Jiang, Tao; Deng, Meng; Nair, Lakshmi S; Khan, Yusuf M; Laurencin, Cato T

2007-12-01

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.

Phase Transitions in a Model of Y-Molecules Abstract

NASA Astrophysics Data System (ADS)

Holz, Danielle; Ruth, Donovan; Toral, Raul; Gunton, James

Immunoglobulin is a Y-shaped molecule that functions as an antibody to neutralize pathogens. In special cases where there is a high concentration of immunoglobulin molecules, self-aggregation can occur and the molecules undergo phase transitions. This prevents the molecules from completing their function. We used a simplified model of 2-Dimensional Y-molecules with three identical arms on a triangular lattice with 2-dimensional Grand Canonical Ensemble. The molecules were permitted to be placed, removed, rotated or moved on the lattice. Once phase coexistence was found, we used histogram reweighting and multicanonical sampling to calculate our phase diagram.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro.

PubMed

Baillie-Johnson, Peter; van den Brink, Susanne Carina; Balayo, Tina; Turner, David Andrew; Martinez Arias, Alfonso

2015-11-24

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

How Nucleus Mechanics and ECM Microstructure Influence the Invasion of Single Cells and Multicellular Aggregates.

PubMed

Giverso, Chiara; Arduino, Alessandro; Preziosi, Luigi

2018-05-01

In order to move in a three-dimensional extracellular matrix, the nucleus of a cell must squeeze through the narrow spacing among the fibers and, by adhering to them, the cell needs to exert sufficiently strong traction forces. If the nucleus is too stiff, the spacing too narrow, or traction forces too weak, the cell is not able to penetrate the network. In this article, we formulate a mathematical model based on an energetic approach, for cells entering cylindrical channels composed of extracellular matrix fibers. Treating the nucleus as an elastic body covered by an elastic membrane, the energetic balance leads to the definition of a necessary criterion for cells to pass through the regular network of fibers, depending on the traction forces exerted by the cells (or possibly passive stresses), the stretchability of the nuclear membrane, the stiffness of the nucleus, and the ratio of the pore size within the extracellular matrix with respect to the nucleus diameter. The results obtained highlight the importance of the interplay between mechanical properties of the cell and microscopic geometric characteristics of the extracellular matrix and give an estimate for a critical value of the pore size that represents the physical limit of migration and can be used in tumor growth models to predict their invasive potential in thick regions of ECM.

Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates

PubMed Central

Yamagishi, Jumpei F; Saito, Nen; Kaneko, Kunihiko

2016-01-01

As cells grow and divide under a given environment, they become crowded and resources are limited, as seen in bacterial biofilms and multicellular aggregates. These cells often show strong interactions through exchanging chemicals, as evident in quorum sensing, to achieve mutualism and division of labor. Here, to achieve stable division of labor, three characteristics are required. First, isogenous cells differentiate into several types. Second, this aggregate of distinct cell types shows better growth than that of isolated cells without interaction and differentiation, by achieving division of labor. Third, this cell aggregate is robust with respect to the number distribution of differentiated cell types. Indeed, theoretical studies have thus far considered how such cooperation is achieved when the ability of cell differentiation is presumed. Here, we address how cells acquire the ability of cell differentiation and division of labor simultaneously, which is also connected with the robustness of a cell society. For this purpose, we developed a dynamical-systems model of cells consisting of chemical components with intracellular catalytic reaction dynamics. The reactions convert external nutrients into internal components for cellular growth, and the divided cells interact through chemical diffusion. We found that cells sharing an identical catalytic network spontaneously differentiate via induction from cell-cell interactions, and then achieve division of labor, enabling a higher growth rate than that in the unicellular case. This symbiotic differentiation emerged for a class of reaction networks under the condition of nutrient limitation and strong cell-cell interactions. Then, robustness in the cell type distribution was achieved, while instability of collective growth could emerge even among the cooperative cells when the internal reserves of products were dominant. The present mechanism is simple and general as a natural consequence of interacting cells with limited resources, and is consistent with the observed behaviors and forms of several aggregates of unicellular organisms. PMID:27749898

Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

DOEpatents

An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

2000-01-01

A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

Functional Human Podocytes Generated in Organoids from Amniotic Fluid Stem Cells

PubMed Central

Benedetti, Valentina; Novelli, Rubina; Abbate, Mauro; Rizzo, Paola; Conti, Sara; Tomasoni, Susanna; Corna, Daniela; Pozzobon, Michela; Cavallotti, Daniela; Yokoo, Takashi; Morigi, Marina; Benigni, Ariela; Remuzzi, Giuseppe

2016-01-01

Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research. PMID:26516208

Collective behavior in two-dimensional biological systems: Receptor clustering and beta-sheet aggregation

NASA Astrophysics Data System (ADS)

Guo, Chinlin

We studied two particular biomedical systems which exhibit collective molecular behavior. One is clustering of tumor necrosis factor receptor I (TNFR1), and another is β-sheet folding and aggregation. Receptor clustering has been shown to be a crucial step in many signaling events but its biological meaning has not been adequately addressed. Here, via a simple lattice model, we show how cells use this clustering machinery to enhance sensitivity as well as robustness. On the other hand, intracellular deposition of aggregated protein rich in β-sheet is a prominent cytopathological feature of most neurodegenerative diseases. How this aggregation occurs and how it responds to therapy is not completely understood. Here, we started from a reconstruction of the H-bond potential and carry out a full investigation of β-sheet thermodynamics as well as kinetics. We show that β-sheet aggregation is most likely due to molecular stacking and found that the minimal length of an aggregate mutant polymer corresponds well with the number observed in adult Huntington's disease. We have also shown that molecular agents such as dendrimers might fail at high-dose therapy; instead, a potential therapy strategy is to block β-turn formation. Our predictions can be used for future experimental tests and clinical trials.

Digital microfluidics for automated hanging drop cell spheroid culture.

PubMed

Aijian, Andrew P; Garrell, Robin L

2015-06-01

Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiologically relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liquid handling, and a wide range of in situ sample preparation and analysis capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. © 2014 Society for Laboratory Automation and Screening.

3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

PubMed

Kucukgul, Can; Ozler, S Burce; Inci, Ilyas; Karakas, Ezgi; Irmak, Ster; Gozuacik, Devrim; Taralp, Alpay; Koc, Bahattin

2015-04-01

Cardiovascular diseases are the leading cause of deaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissue engineering investigations have been based on the cell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, and inflammation of biomaterials used, the recent tissue engineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macrovascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computational algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct. © 2014 Wiley Periodicals, Inc.

The Effect of Different Mixing Methods on Working Time, Setting Time, Dimensional Changes and Film Thickness of Mineral Trioxide Aggregate and Calcium-Enriched Mixture.

PubMed

Shahi, Shahriar; Ghasemi, Negin; Rahimi, Saeed; Yavari, Hamidreza; Janani, Maryam; Mokhtari, Hadi; Bahari, Mahmood; Rabbani, Parastu

2015-01-01

The aim of the present study was to evaluate the effect of different mixing techniques (conventional, amalgamator and ultrasonic mixing) on the physical properties the working time (WT), setting time (ST), dimensional changes (DC) and film thickness (FT)] of calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA). The mentioned physical properties were determined using the ISO 6786:2001 specification. Six samples of each material were prepared for three mixing techniques (totally 36 samples). Data were analyzed using descriptive statistics, two-way ANOVA and Post Hoc Tukey's tests. The level of significance was defined at 0.05. Irrespective of mixing technique, there was no significant difference between the WT and FT of the tested materials. Except for the DC of MTA and the FT of the all materials, other properties were significantly affected with mixing techniques (P<0.05). The ultrasonic technique decreased the ST of MTA and CEM cement and increased the WT of CEM cement (P<0.05). The mixing technique of the materials had no significant effect on the dimensional changes of MTA and the film thickness of both materials.

Cornea and ocular lens visualized with three-dimensional confocal microscopy

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1992-08-01

This paper demonstrates the advantages of three-dimensional reconstruction of the cornea and the ocular crystalline lens by confocal microscopy and volume rendering computer techniques. The advantages of noninvasive observation of ocular structures in living, unstained, unfixed tissue include the following: the tissue is in a natural living state without the artifacts of fixation, mechanical sectioning, and staining; the three-dimensional structure can be observed from any view point and quantitatively analyzed; the dynamics of morphological changes can be studied; and the use of confocal microscopic observation results in a reduction of the number of animals required for ocular morphometric studies. The main advantage is that the dynamic morphology of ocular structures can be investigated in living ocular tissue. A laser scanning confocal microscope was used in the reflected light mode to obtain the two- dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with 488 nm wavelength. The microscope objective was a Leitz 25X, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The under sampling resulted in a three-dimensional visualization rendering in which the corneal thickness (z-axis) is compressed. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their `beaded' cell borders, basal lamina, nerve plexus, nerve fibers, free nerve endings in the basal epithelial cells, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in-situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers.

Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

PubMed

Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

2015-12-01

Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P < 0.05), respectively. Epifluorescence microscopy showed that the proportion of blastocysts with eGFP-positive cells in the ICM was higher in the PHA group than in the no-PHA group (40% vs. 16%; P < 0.05). Confocal laser scanning microscopy revealed that the total cell numbers of blastocysts from the PHA group of aggregation chimeras (n = 17; 207.8 ± 67.3 [mean ± standard deviation]) were higher (P < 0.05) than those of embryos without ZP and exposed to PHA (n = 30; 159.6 ± 42.2) and of handling control embryos (n = 19; 176.9 ± 53.3). The same was true for ICM cell counts (56.5 ± 22.0 vs. 37.7 ± 14.2 and 38.7 ± 12.4) and TE cell counts (151.2 ± 58.0 vs. 121.9 ± 37.4 and 138.3 ± 53.0), whereas the ICM/total cell number ratio was not different between the groups. Of the 17 chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescent transgenic mice suitable for multi-color aggregation chimera studies.

PubMed

Ohtsuka, Masato; Miura, Hiromi; Gurumurthy, Channabasavaiah B; Kimura, Minoru; Inoko, Hidetoshi; Yoshimura, Shinichi; Sato, Masahiro

2012-11-01

We recently reported a novel method of mouse transgenesis called Pronuclear Injection-based Targeted Transgenisis (PITT) using which a series of fluorescent transgenic (Tg) mice lines were generated. These lines, unlike those generated using conventional random integration methods, express the transgenes faithfully and reproducibly generation after generation. Because of this superior nature, these lines are ideal for the generation of multi-colored aggregation chimeras that can be used to study cell-cell interactions and lineage analyses in living embryos/organs, where the transgenes can be detected and the clonal origin of a given cell population easily traced by its distinct fluorescence. In this study, to verify if Tg fluorescent mice generated through PITT were suitable for such applications, we sought to generate chimeric blastocysts and chimeric-Tg mice by aggregating two- or three-colored 8-cell embryos. Our analyses using these models led to the following observations. First, we noticed that cell mixing was infrequent during the stages of morula to early blastocyst. Second, chimeric fetuses obtained after aggregation of the two-colored 8-cell embryos exhibited uniform cell mixing. And third, in the organs of adult chimeric mice, the mode of cell distribution could be either clonal or polyclonal, as previously pointed out by others. Implications of our novel and improved Tg-chimeric mice approach for clonal cell lineage and developmental studies are discussed.

Some observations on the three-dimensional growth of L5178Y cell colonies in soft agar culture.

NASA Technical Reports Server (NTRS)

Dalen, H.; Burki, H. J.

1971-01-01

The three-dimensional organization of spherical colonies formed by L5178Y cells grown in soft agar cultures was investigated by light and scanning electron microscopy. Visible colonies were formed after 7 days of incubation and increased in size for more than 2 weeks. At this time the colonies contained a central core of necrotic cells surrounded by an outer shell of normal-looking cells in loose contact with each other. Cross sectional radioautographs revealed that tritiated precursors were incorporated only into those cells in the ?viable cell' shell and not in the necrotic center of the colony. It is pointed out that increased knowledge of the factors leading to this type of three-dimensional organization is of particular interest, since it is similar to the conditions found in certain types of solid tumors (Thomlinson and Gray, 1955).

Ultra-high-Q three-dimensional photonic crystal nano-resonators.

PubMed

Tang, Lingling; Yoshie, Tomoyuki

2007-12-10

Two nano-resonator modes are designed in a woodpile three-dimensional photonic crystal by the modulation of unit cell size along a low-loss optical waveguide. One is a dipole mode with 2.88 cubic half-wavelengths mode volume. The other is a quadrupole mode with 8.3 cubic half-wavelengths mode volume. Light is three-dimensionally confined by a complete photonic band gap so that, in the analyzed range, the quality factor exponentially increases as the increase in the number of unit cells used for confinement of light.

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

NASA Astrophysics Data System (ADS)

Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

2005-05-01

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

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

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

PubMed

Moro, L N; Jarazo, J; Buemo, C; Hiriart, M I; Sestelo, A; Salamone, D F

2015-10-01

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation. © 2015 Blackwell Verlag GmbH.

Three-dimensional morphological imaging of human induced pluripotent stem cells by using low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Kakuno, Yumi; Goto, Kentaro; Fukami, Tadashi; Sugiyama, Norikazu; Iwai, Hidenao; Mizuguchi, Yoshinori; Yamashita, Yutaka

2014-03-01

There is an increasing need for non-invasive imaging techniques in the field of stem cell research. Label-free techniques are the best choice for assessment of stem cells because the cells remain intact after imaging and can be used for further studies such as differentiation induction. To develop a high-resolution label-free imaging system, we have been working on a low-coherence quantitative phase microscope (LC-QPM). LC-QPM is a Linnik-type interference microscope equipped with nanometer-resolution optical-path-length control and capable of obtaining three-dimensional volumetric images. The lateral and vertical resolutions of our system are respectively 0.5 and 0.93 μm and this performance allows capturing sub-cellular morphological features of live cells without labeling. Utilizing LC-QPM, we reported on three-dimensional imaging of membrane fluctuations, dynamics of filopodia, and motions of intracellular organelles. In this presentation, we report three-dimensional morphological imaging of human induced pluripotent stem cells (hiPS cells). Two groups of monolayer hiPS cell cultures were prepared so that one group was cultured in a suitable culture medium that kept the cells undifferentiated, and the other group was cultured in a medium supplemented with retinoic acid, which forces the stem cells to differentiate. The volumetric images of the 2 groups show distinctive differences, especially in surface roughness. We believe that our LC-QPM system will prove useful in assessing many other stem cell conditions.

Emergent dynamics of laboratory insect swarms

NASA Astrophysics Data System (ADS)

Kelley, Douglas H.; Ouellette, Nicholas T.

2013-01-01

Collective animal behaviour occurs at nearly every biological size scale, from single-celled organisms to the largest animals on earth. It has long been known that models with simple interaction rules can reproduce qualitative features of this complex behaviour. But determining whether these models accurately capture the biology requires data from real animals, which has historically been difficult to obtain. Here, we report three-dimensional, time-resolved measurements of the positions, velocities, and accelerations of individual insects in laboratory swarms of the midge Chironomus riparius. Even though the swarms do not show an overall polarisation, we find statistical evidence for local clusters of correlated motion. We also show that the swarms display an effective large-scale potential that keeps individuals bound together, and we characterize the shape of this potential. Our results provide quantitative data against which the emergent characteristics of animal aggregation models can be benchmarked.

Evaluation of the metabolic capability of primary human hepatocytes in three-dimensional cultures on microstructural plates.

PubMed

Koyama, Satoshi; Arakawa, Hiroshi; Itoh, Manabu; Masuda, Norio; Yano, Kentaro; Kojima, Hajime; Ogihara, Takuo

2018-04-01

The NanoCulture Plate (NCP) is a novel microstructural plate designed as a base for the three-dimensional culture of cells/tissues. This study examined whether or not the metabolic capability of human primary hepatocytes is well maintained during culture on NCPs. The hepatocytes formed aggregates after seeding and their ATP content was well maintained during culture for 21 days. Expression of CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 mRNAs was detected throughout the 21-day culture period. Addition of CYP substrate drugs (midazolam, diclofenac, lamotrigine and acetaminophen) resulted in the formation of multiple metabolites with a corresponding decrease in the amounts of the unchanged compounds. The inducers omeprazole, phenobarbital and rifampicin increased the levels of CYP1A2, 2B6 and 3A4 mRNAs by 110-fold, 12.5-fold and 5.4-fold, respectively, at day 2, compared with control human hepatocytes. CYP activities were also increased at 2 days after inducer treatment (CYP1A2, 2.2-fold; CYP2B6, 20.6-fold; CYP3A4, 3.3-fold). The results indicate that the hepatocyte spheroids on NCP have detectable and inducible metabolic abilities during the 7-day culture period. Copyright © 2018 John Wiley & Sons, Ltd.

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

PubMed

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

2011-01-01

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

Improved function and growth of pancreatic cells in a three-dimensional bioreactor environment.

PubMed

Samuelson, Lisa; Gerber, David A

2013-01-01

Methods of three-dimensional (3D) cell culture have made significant progress in recent years due to a better understanding of cell to cell interactions and the cell's interface with their surrounding environment. We hypothesized that a microgravity 3D culture system would improve upon the growth and function of a pancreatic progenitor cell population. We developed a rotating wall vessel bioreactor and established a culture system using a pancreatic cell line. Cells in the bioreactors showed robust proliferation, enhanced transcriptional signaling, and improved translation of pancreatic genes compared with two-dimensional static culture. Cells also gained the ability to respond to glucose stimulation, which was not observed in the control cultures. These findings suggest that a 3D microgravity bioreactor environment mimics the niche of the pancreas yielding a cell source with potential for cell-based therapy in the treatment of diabetes.

Dynamic chromosomal rearrangements in Hodgkin's lymphoma are due to ongoing three-dimensional nuclear remodeling and breakage-bridge-fusion cycles.

PubMed

Guffei, Amanda; Sarkar, Rahul; Klewes, Ludger; Righolt, Christiaan; Knecht, Hans; Mai, Sabine

2010-12-01

Hodgkin's lymphoma is characterized by the presence of mono-nucleated Hodgkin cells and bi- to multi-nucleated Reed-Sternberg cells. We have recently shown telomere dysfunction and aberrant synchronous/asynchronous cell divisions during the transition of Hodgkin cells to Reed-Sternberg cells.1 To determine whether overall changes in nuclear architecture affect genomic instability during the transition of Hodgkin cells to Reed-Sternberg cells, we investigated the nuclear organization of chromosomes in these cells. Three-dimensional fluorescent in situ hybridization revealed irregular nuclear positioning of individual chromosomes in Hodgkin cells and, more so, in Reed-Sternberg cells. We characterized an increasingly unequal distribution of chromosomes as mono-nucleated cells became multi-nucleated cells, some of which also contained chromosome-poor 'ghost' cell nuclei. Measurements of nuclear chromosome positions suggested chromosome overlaps in both types of cells. Spectral karyotyping then revealed both aneuploidy and complex chromosomal rearrangements: multiple breakage-bridge-fusion cycles were at the origin of the multiple rearranged chromosomes. This conclusion was challenged by super resolution three-dimensional structured illumination imaging of Hodgkin and Reed-Sternberg nuclei. Three-dimensional super resolution microscopy data documented inter-nuclear DNA bridges in multi-nucleated cells but not in mono-nucleated cells. These bridges consisted of chromatids and chromosomes shared by two Reed-Sternberg nuclei. The complexity of chromosomal rearrangements increased as Hodgkin cells developed into multi-nucleated cells, thus indicating tumor progression and evolution in Hodgkin's lymphoma, with Reed-Sternberg cells representing the highest complexity in chromosomal rearrangements in this disease. This is the first study to demonstrate nuclear remodeling and associated genomic instability leading to the generation of Reed-Sternberg cells of Hodgkin's lymphoma. We defined nuclear remodeling as a key feature of Hodgkin's lymphoma, highlighting the relevance of nuclear architecture in cancer.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor.

PubMed

Ingram, M; Techy, G B; Saroufeem, R; Yazan, O; Narayan, K S; Goodwin, T J; Spaulding, G F

1997-06-01

Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated. The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture. Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm. Architecture of spheroids varied with cell type. Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures. Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs. Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells. Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

A Structure-Based Distance Metric for High-Dimensional Space Exploration with Multi-Dimensional Scaling

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

Lee, Hyun Jung; McDonnell, Kevin T.; Zelenyuk, Alla

2014-03-01

Although the Euclidean distance does well in measuring data distances within high-dimensional clusters, it does poorly when it comes to gauging inter-cluster distances. This significantly impacts the quality of global, low-dimensional space embedding procedures such as the popular multi-dimensional scaling (MDS) where one can often observe non-intuitive layouts. We were inspired by the perceptual processes evoked in the method of parallel coordinates which enables users to visually aggregate the data by the patterns the polylines exhibit across the dimension axes. We call the path of such a polyline its structure and suggest a metric that captures this structure directly inmore » high-dimensional space. This allows us to better gauge the distances of spatially distant data constellations and so achieve data aggregations in MDS plots that are more cognizant of existing high-dimensional structure similarities. Our MDS plots also exhibit similar visual relationships as the method of parallel coordinates which is often used alongside to visualize the high-dimensional data in raw form. We then cast our metric into a bi-scale framework which distinguishes far-distances from near-distances. The coarser scale uses the structural similarity metric to separate data aggregates obtained by prior classification or clustering, while the finer scale employs the appropriate Euclidean distance.« less

Three-dimensional cell to tissue assembly process

NASA Technical Reports Server (NTRS)

Wolf, David A. (Inventor); Schwarz, Ray P. (Inventor); Lewis, Marian L. (Inventor); Cross, John H. (Inventor); Huls, Mary H. (Inventor)

1992-01-01

The present invention relates a 3-dimensional cell to tissue and maintenance process, more particularly to methods of culturing cells in a culture environment, either in space or in a gravity field, with minimum fluid shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region.

Utilize Cementitious High Carbon Fly Ash (CHCFA) to Stabilize Cold In-Place Recycled (CIR) Asphalt Pavement as Base Coarse

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

Wen, Haifang; Li, Xiaojun; Edil, Tuncer

The purpose of this study was to evaluate the performance of cementitious high carbon fly ash (CHCFA) stabilized recycled asphalt pavement as a base course material in a real world setting. Three test road cells were built at MnROAD facility in Minnesota. These cells have the same asphalt surface layers, subbases, and subgrades, but three different base courses: conventional crushed aggregates, untreated recycled pavement materials (RPM), and CHCFA stabilized RPM materials. During and after the construction of the three cells, laboratory and field tests were carried out to characterize the material properties. The test results were used in the mechanistic-empiricalmore » pavement design guide (MEPDG) to predict the pavement performance. Based on the performance prediction, the life cycle analyses of cost, energy consumption, and greenhouse gasses were performed. The leaching impacts of these three types of base materials were compared. The laboratory and field tests showed that fly ash stabilized RPM had higher modulus than crushed aggregate and RPM did. Based on the MEPDG performance prediction, the service life of the Cell 79 containing fly ash stabilized RPM, is 23.5 years, which is about twice the service life (11 years) of the Cell 77 with RPM base, and about three times the service life (7.5 years) of the Cell 78 with crushed aggregate base. The life cycle analysis indicated that the usage of the fly ash stabilized RPM as the base of the flexible pavement can significantly reduce the life cycle cost, the energy consumption, the greenhouse gases emission. Concentrations of many trace elements, particularly those with relatively low water quality standards, diminish over time as water flows through the pavement profile. For many elements, concentrations below US water drinking water quality standards are attained at the bottom of the pavement profile within 2-4 pore volumes of flow.« less

Differential toxicity, conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids.

PubMed

Galante, Denise; Corsaro, Alessandro; Florio, Tullio; Vella, Serena; Pagano, Aldo; Sbrana, Francesca; Vassalli, Massimo; Perico, Angelo; D'Arrigo, Cristina

2012-11-01

Among the different species of water-soluble β-peptides (Aβ1-42, Aβ1-40 and N-terminal truncated Aβ-peptides), Aβpy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aβpy3-42, we separated different aggregation states of Aβ1-42 and Aβpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical β-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given β-peptide and among the aggregate of the two β-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aβ1-42 the most toxic aggregate is la, while in the case of Aβpy3-42 both sa and la are equally toxic. Aβ aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aβpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aβpy3-42 peptide. Copyright © 2012 Elsevier Ltd. All rights reserved.

Quantification of three-dimensional cell-mediated collagen remodeling using graph theory.

PubMed

Bilgin, Cemal Cagatay; Lund, Amanda W; Can, Ali; Plopper, George E; Yener, Bülent

2010-09-30

Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Definition of early metrics that are able to predict long-term functionality by linking engineered tissue structure to function is an important step toward optimizing biomaterials for the purposes of regenerative medicine.

Solvent induced modifications to fiber nanostructure and morphology for 12HSA molecular gels

NASA Astrophysics Data System (ADS)

Gao, Jie

Molecular organogels are thermo reversible quasi-solid materials, which are formed by low molecular weight organogelators (LMOGs) undergoing supramolecular aggregation via non-covalent interactions, forming a three-dimensional fibrillar network. Numerous applications of molecular organogels are been investigated as edible oils, drug release matrices and personal care products. The chemistry of the organic phase (i.e., solvent) influences every level of structure in organogels. Different solvents induce LMOG to assemble into "crystal like" fibers, which have more than one crystal form, lamellar arrangement and domain size. Differences in these solid states are known to affect the macroscopic properties of the gel, including critical gelator concentration (CGC), melting point, melting enthalpy and opacity.12-hydroxystearic acid (12HSA) was examined in several classes of organic solvents with different function groups. These gels, sols or precipitates were analyzed using a series of techniques including: powder x-ray diffraction (XRD), differential scanning calorimetry (DSC), fourier-transform infrared spectroscopy (FT-IR), pulsed nuclear magnetic resonance spectroscopy (pNMR) and microscopy. Specifically, certain solvents caused 12HSA to self-assemble into a triclinic parallel polymorphic form with subcell spacing of ~4.6, 3.9, and 3.8 A and an interdigitated unit cell with a lamellar arrangement (38~44 A). This polymorphic form corresponded to a less effective sphereultic supramolecular crystalline network, which immobilizes solvents at CGC greater than 1.5 wt %. The other group of solvents induce a hexagonal subcell spacing (i.e., unit sub cell spacing ~4.1 A) and are arranged in a multi lamellar fashion with a unit cell greater than the bimolecular length of 12HSA (~54 A).This polymorphic form corresponds to fibrillar aggregates with a CGC less than 1 wt %.

Assembly of Triblock Amphiphilic Peptides into One-Dimensional Aggregates and Network Formation.

PubMed

Ozgur, Beytullah; Sayar, Mehmet

2016-10-06

Peptide assembly plays a key role in both neurological diseases and development of novel biomaterials with well-defined nanostructures. Synthetic model peptides provide a unique platform to explore the role of intermolecular interactions in the assembly process. A triblock peptide architecture designed by the Hartgerink group is a versatile system which relies on Coulomb interactions, hydrogen bonding, and hydrophobicity to guide these peptides' assembly at three different length scales: β-sheets, double-wall ribbon-like aggregates, and finally a highly porous network structure which can support gels with ≤1% by weight peptide concentration. In this study, by using molecular dynamics simulations of a structure based implicit solvent coarse grained model, we analyzed this hierarchical assembly process. Parametrization of our CG model is based on multiple-state points from atomistic simulations, which enables this model to represent the conformational adaptability of the triblock peptide molecule based on the surrounding medium. Our results indicate that emergence of the double-wall β-sheet packing mechanism, proposed in light of the experimental evidence, strongly depends on the subtle balance of the intermolecular forces. We demonstrate that, even though backbone hydrogen bonding dominates the early nucleation stages, depending on the strength of the hydrophobic and Coulomb forces, alternative structures such as zero-dimensional aggregates with two β-sheets oriented orthogonally (which we refer to as a cross-packed structure) and β-sheets with misoriented hydrophobic side chains are also feasible. We discuss the implications of these competing structures for the three different length scales of assembly by systematically investigating the influence of density, counterion valency, and hydrophobicity.

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

PubMed

Coppen, S R; Newsam, R; Bull, A T; Baines, A J

1995-04-20

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

Magnetic levitating polymeric nano/microparticular substrates for three-dimensional tumor cell culture.

PubMed

Lee, Woong Ryeol; Oh, Kyung Taek; Park, So Young; Yoo, Na Young; Ahn, Yong Sik; Lee, Don Haeng; Youn, Yu Seok; Lee, Deok-Keun; Cha, Kyung-Hoi; Lee, Eun Seong

2011-07-01

Herein, we describe magnetic cell levitation models using conventional polymeric microparticles or nanoparticles as a substrate for the three-dimensional tumor cell culture. When the magnetic force originating from the ring-shaped magnets overcame the gravitational force, the magnetic field-levitated KB tumor cells adhered to the surface area of magnetic iron oxide (Fe(3)O(4))-encapsulated nano/microparticles and concentrated clusters of levitated cells, ultimately developing tumor cells to tumor spheroids. These simple cell culture models may prove useful for the screening of anticancer drugs and their formulations. Copyright © 2011 Elsevier B.V. All rights reserved.

Cell culture for three-dimensional modeling in rotating-wall vessels: an application of simulated microgravity

NASA Technical Reports Server (NTRS)

Schwarz, R. P.; Goodwin, T. J.; Wolf, D. A.

1992-01-01

High-density, three-dimensional cell cultures are difficult to grow in vitro. The rotating-wall vessel (RWV) described here has cultured BHK-21 cells to a density of 1.1 X 10(7) cells/ml. Cells on microcarriers were observed to grow with enhanced bridging in this batch culture system. The RWV is a horizontally rotated tissue culture vessel with silicon membrane oxygenation. This design results in a low-turbulence, low-shear cell culture environment with abundant oxygenation. The RWV has the potential to culture a wide variety of normal and neoplastic cells.

A new three-dimensional manufacturing service composition method under various structures using improved Flower Pollination Algorithm

NASA Astrophysics Data System (ADS)

Zhang, Wenyu; Yang, Yushu; Zhang, Shuai; Yu, Dejian; Chen, Yong

2018-05-01

With the growing complexity of customer requirements and the increasing scale of manufacturing services, how to select and combine the single services to meet the complex demand of the customer has become a growing concern. This paper presents a new manufacturing service composition method to solve the multi-objective optimization problem based on quality of service (QoS). The proposed model not only presents different methods for calculating the transportation time and transportation cost under various structures but also solves the three-dimensional composition optimization problem, including service aggregation, service selection, and service scheduling simultaneously. Further, an improved Flower Pollination Algorithm (IFPA) is proposed to solve the three-dimensional composition optimization problem using a matrix-based representation scheme. The mutation operator and crossover operator of the Differential Evolution (DE) algorithm are also used to extend the basic Flower Pollination Algorithm (FPA) to improve its performance. Compared to Genetic Algorithm, DE, and basic FPA, the experimental results confirm that the proposed method demonstrates superior performance than other meta heuristic algorithms and can obtain better manufacturing service composition solutions.

Growing Three-Dimensional Cartilage-Cell Cultures

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F.; Prewett, Tacey L.; Goodwin, Thomas J.

1995-01-01

Process for growing three-dimensional cultures of mammalian cartilage from normal mammalian cells devised. Effected using horizontal rotating bioreactor described in companion article, "Simplified Bioreactor for Growing Mammalian Cells" (MSC-22060). Bioreactor provides quiescent environment with generous supplies of nutrient and oxygen. Initiated with noncartilage cells. Artificially grown tissue resembles that in mammalian cartilage. Potential use in developing therapies for damage to cartilage by joint and back injuries and by such inflammatory diseases as arthritis and temporal-mandibular joint disease. Also used to test nonsteroid anti-inflammation medicines.

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

PubMed Central

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

2014-01-01

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

Computational cell quantification in the human brain tissues based on hard x-ray phase-contrast tomograms

NASA Astrophysics Data System (ADS)

Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Chicherova, Natalia; Rack, Alexander; Zdora, Marie-Christine; Zanette, Irene; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

2016-10-01

Cell visualization and counting plays a crucial role in biological and medical research including the study of neurodegenerative diseases. The neuronal cell loss is typically determined to measure the extent of the disease. Its characterization is challenging because the cell density and size already differs by more than three orders of magnitude in a healthy cerebellum. Cell visualization is commonly performed by histology and fluorescence microscopy. These techniques are limited to resolve complex microstructures in the third dimension. Phase- contrast tomography has been proven to provide sufficient contrast in the three-dimensional imaging of soft tissue down to the cell level and, therefore, offers the basis for the three-dimensional segmentation. Within this context, a human cerebellum sample was embedded in paraffin and measured in local phase-contrast mode at the beamline ID19 (ESRF, Grenoble, France) and the Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK). After the application of Frangi-based filtering the data showed sufficient contrast to automatically identify the Purkinje cells and to quantify their density to 177 cells per mm3 within the volume of interest. Moreover, brain layers were segmented in a region of interest based on edge detection. Subsequently performed histological analysis validated the presence of the cells, which required a mapping from the two- dimensional histological slices to the three-dimensional tomogram. The methodology can also be applied to further tissue types and shows potential for the computational tissue analysis in health and disease.

Three-Dimensional Transgenic Cell Models to Quantify Space Genotoxic Effects

NASA Technical Reports Server (NTRS)

Gonda, S.; Wu, H.; Pingerelli, P.; Glickman, B.

2000-01-01

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of mUltiple copies of defined target genes for genotoxic assessment. The Rat 2(lambda) fibroblasts (Stratagene, Inc.) were genetically engineered to contain high-density target genes for mutagenesis. Stable three-dimensional, multicellular spheroids were formed when human mammary epithelial cells and Rat 2(lambda) fibroblasts were cocultured on Cytodex 3 Beads in a rotating wall bioreactor. The utility of this spheroidal model for genotoxic assessment was indicated by a linear dose response curve and by results of gene sequence analysis of mutant clones from 400micron diameter spheroids following low-dose, high-energy, neon radiation exposure

Clonorchis sinensis excretory-secretory products promote the migration and invasion of cholangiocarcinoma cells by activating the integrin β4-FAK/Src signaling pathway.

PubMed

Pak, Jhang Ho; Bashir, Qudsia; Kim, In Ki; Hong, Sung-Jong; Maeng, Sejung; Bahk, Young Yil; Kim, Tong-Soo

2017-06-01

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression. Copyright © 2017 Elsevier B.V. All rights reserved.

An HCG-rich microenvironment contributes to ovarian cancer cell differentiation into endothelioid cells in a three-dimensional culture system.

PubMed

Su, Min; Fan, Chao; Gao, Sainan; Shen, Aiguo; Wang, Xiaoying; Zhang, Yuquan

2015-11-01

We investigated the expression of human chorionic gonadotropin (HCG) and its effects on vasculogenic mimicry (VM) formation in ovarian cancer cells under normoxic and hypoxic conditions in three-dimensional matrices preconditioned by an endothelial-trophoblast cell co-culture system. The co-culture model was established using human umbilical vein endothelial cells (HUVECs) and HTR-8 trophoblast cells in a three-dimensional culture system. The co-cultured cells were removed with NH4OH, and ovarian cancer cells were implanted into the preconditioned matrix. VM was identified morphologically and by detecting vascular markers expressed by cancer cells. The specificity of the effects of exogenous HCG in the microenvironment was assessed by inhibition with a neutralizing anti-HCG antibody. HCG siRNA was used to knock down endogenous HCG expression in OVCAR-3 ovarian cancer cells. HTR-8 cells 'fingerprinted' HUVECs to form capillary-like tube structures in co-cultures. In the preconditioned HCG-rich microenvironment, the number of vessel-like network structures formed by HCG receptor-positive OVCAR-3 cells and the expression levels of CD31, VEGF and factor VIII were significantly increased. The preconditioned HCG-rich microenvironment significantly increased the expression of hypoxia inducible factor-1α (HIF‑1α) and VM formation in OVCAR-3 cells under hypoxic conditions. Treatment with a neutralizing anti-HCG antibody but not HCG siRNA significantly inhibited the formation of vessel-like network structures. HCG in the microenvironment contributes to OVCAR-3 differentiation into endothelioid cells in three-dimensional matrices preconditioned with an endothelial-trophoblast cell co-culture system. HCG may synergistically enhance hypoxia-induced vascular markers and HIF-1α expression. These findings would provide perspectives on new therapeutic targets for ovarian cancer.

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

PubMed

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

A three-dimensional neural spheroid model for capillary-like network formation.

PubMed

Boutin, Molly E; Kramer, Liana L; Livi, Liane L; Brown, Tyler; Moore, Christopher; Hoffman-Kim, Diane

2018-04-01

In vitro three-dimensional neural spheroid models have an in vivo-like cell density, and have the potential to reduce animal usage and increase experimental throughput. The aim of this study was to establish a spheroid model to study the formation of capillary-like networks in a three-dimensional environment that incorporates both neuronal and glial cell types, and does not require exogenous vasculogenic growth factors. We created self-assembled, scaffold-free cellular spheroids using primary-derived postnatal rodent cortex as a cell source. The interactions between relevant neural cell types, basement membrane proteins, and endothelial cells were characterized by immunohistochemistry. Transmission electron microscopy was used to determine if endothelial network structures had lumens. Endothelial cells within cortical spheroids assembled into capillary-like networks with lumens. Networks were surrounded by basement membrane proteins, including laminin, fibronectin and collagen IV, as well as key neurovascular cell types. Existing in vitro models of the cortical neurovascular environment study monolayers of endothelial cells, either on transwell inserts or coating cellular spheroids. These models are not well suited to study vasculogenesis, a process hallmarked by endothelial cell cord formation and subsequent lumenization. The neural spheroid is a new model to study the formation of endothelial cell capillary-like structures in vitro within a high cell density three-dimensional environment that contains both neuronal and glial populations. This model can be applied to investigate vascular assembly in healthy or disease states, such as stroke, traumatic brain injury, or neurodegenerative disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Coherent three-dimensional X-ray cryo-imaging.

PubMed

Robinson, Ian

2015-09-01

The combination of cryogenic sample temperatures with three-dimensional coherent diffractive imaging for the case of whole frozen-hydrated cells is discussed in the light of theoretical predictions of the achievable resolution.

Large three-dimensional photonic crystals based on monocrystalline liquid crystal blue phases.

PubMed

Chen, Chun-Wei; Hou, Chien-Tsung; Li, Cheng-Chang; Jau, Hung-Chang; Wang, Chun-Ta; Hong, Ching-Lang; Guo, Duan-Yi; Wang, Cheng-Yu; Chiang, Sheng-Ping; Bunning, Timothy J; Khoo, Iam-Choon; Lin, Tsung-Hsien

2017-09-28

Although there have been intense efforts to fabricate large three-dimensional photonic crystals in order to realize their full potential, the technologies developed so far are still beset with various material processing and cost issues. Conventional top-down fabrications are costly and time-consuming, whereas natural self-assembly and bottom-up fabrications often result in high defect density and limited dimensions. Here we report the fabrication of extraordinarily large monocrystalline photonic crystals by controlling the self-assembly processes which occur in unique phases of liquid crystals that exhibit three-dimensional photonic-crystalline properties called liquid-crystal blue phases. In particular, we have developed a gradient-temperature technique that enables three-dimensional photonic crystals to grow to lateral dimensions of ~1 cm (~30,000 of unit cells) and thickness of ~100 μm (~ 300 unit cells). These giant single crystals exhibit extraordinarily sharp photonic bandgaps with high reflectivity, long-range periodicity in all dimensions and well-defined lattice orientation.Conventional fabrication approaches for large-size three-dimensional photonic crystals are problematic. By properly controlling the self-assembly processes, the authors report the fabrication of monocrystalline blue phase liquid crystals that exhibit three-dimensional photonic-crystalline properties.

Collective Behavior of Amoebae in Thin Films

NASA Astrophysics Data System (ADS)

Bae, Albert

2005-03-01

We have discovered new aspects of social behavior in Dictyostelium discoideum by culturing high density colonies in liquid media depleted of nutrients in confined geometries by using three different preparations: I. thin (15-40um thick) and II. ultrathin (<3um) films of liquid media with a mineral oil overlayer, and III. microfluidic chambers fabricated in PDMS (˜7um tall). We find greatly reduced, if not eliminated, cell on cell layering in the microfluidic system when compared to the wetting layer preparations. The ultrathin films reveal robust behavior of cells despite flattening that increased their areas by over an order of magnitude. We also observed that the earliest synchronized response of cells following the onset of starvation, a precursor to aggregation, was hastened by reducing the thickness of the aqueous culture layer. We were surprised to find that the threshold concentration for aggregation was raised by thin film confinement when compared to bulk behavior. Finally, both the ultra thin and microfluidic preparations reveal, with new clarity, vortex states of aggregation.

Aggregation and morphology control enables multiple cases of high-efficiency polymer solar cells

PubMed Central

Liu, Yuhang; Zhao, Jingbo; Li, Zhengke; Mu, Cheng; Hu, Huawei; Jiang, Kui; Lin, Haoran; Ade, Harald; Yan, He

2014-01-01

Although the field of polymer solar cell has seen much progress in device performance in the past few years, several limitations are holding back its further development. For instance, current high-efficiency (>9.0%) cells are restricted to material combinations that are based on limited donor polymers and only one specific fullerene acceptor. Here we report the achievement of high-performance (efficiencies up to 10.8%, fill factors up to 77%) thick-film polymer solar cells for multiple polymer:fullerene combinations via the formation of a near-ideal polymer:fullerene morphology that contains highly crystalline yet reasonably small polymer domains. This morphology is controlled by the temperature-dependent aggregation behaviour of the donor polymers and is insensitive to the choice of fullerenes. The uncovered aggregation and design rules yield three high-efficiency (>10%) donor polymers and will allow further synthetic advances and matching of both the polymer and fullerene materials, potentially leading to significantly improved performance and increased design flexibility. PMID:25382026

Formation of wormlike aggregates of fluorocarbon-hydrocarbon hybrid surfactant by Langmuir-Blodgett transfer and alignment of gold nanoparticles.

PubMed

Kondo, Yukishige; Fukuoka, Hiroshi; Nakano, Shuichi; Hayashi, Kohei; Tsukagoshi, Tatsuya; Matsumoto, Mutsuyoshi; Yoshino, Norio

2007-05-22

A novel anionic fluorocarbon-hydrocarbon hybrid surfactant (SS-Hyb-Na+) with a disulfide group has been synthesized from 11-bromo-1-undecanal and perfluorohexylethyl iodide via three steps. The Langmuir-Blodgett (LB) transfer of the 1:100 (mol/mol) mixed monolayer of SS-Hyb-Na+ and stearyl alcohol (C18OH) formed on an aqueous solution containing a cationic polymer, poly(diallyldimethylammonium chloride) (PDDA+Cl-) onto a hydrophobic silicon wafer yields the formation of wormlike aggregates consisting of SS-Hyb-/PDDA+ polyion complexes. It is found that the aggregates align along the withdrawal direction of the wafer substrate. When the wafer on which the wormlike aggregates exist is immersed into the dispersion of gold nanoparticles (Au NPs) prepared by the citrate reduction method, Au NPs align along the wormlike structures. Even though the surface of the wafer is placed either vertical or parallel to the monolayer compression direction during the LB transfer, the one-dimensional (1D) array of Au NPs is observed along the withdrawal direction of the wafer. This indicates that the wormlike aggregates of SS-Hyb-/PDDA+ complexes are aligned during the LB transfer, and the aligned aggregates behave as a scaffold in the 1D array of Au NPs.

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

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

Phosphorylation of hepatocyte growth factor receptor and epidermal growth factor receptor of human hepatocytes can be maintained in a (3D) collagen sandwich culture system.

PubMed

Engl, Tobias; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Jonas, Dietger; Oppermann, Elsie; Auth, Marcus K H; Schaudt, André; Bechstein, Wolf-Otto; Blaheta, Roman A

2004-08-01

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three-dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c-met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two-dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.

Unveiling One-Dimensional Supramolecular Structures Formed through π-π Stacking of Phenothiazines by Differential Pulse Voltammetry.

PubMed

Carvalho, Fernando R; Zampieri, Eduardo H; Caetano, Wilker; Silva, Rafael

2017-05-19

Organic-based nanomaterials can be self-assembled by strong and directional intermolecular forces such as π-π interactions. Experimental information about the stability, size, and geometry of the formed structures is very limited for species that easily aggregate, even at very low concentrations. Differential pulse voltammetry (DPV) can unveil the formation, growth, and also the stability window of ordered, one-dimensional, lamellar self-aggregates formed by supramolecular π stacking of phenothiazines at micromolar (10 -6  mol L -1 ) concentrations. The self-diffusion features of the species at different concentrations are determined by DPV and used to probe the π staking process through the concept of the frictional resistance. It is observed that toluidine blue and methylene blue start to self-aggregate around 9 μmol L -1 , and that the self-aggregation process occurs by one-dimensional growth as the concentration of the phenothiazines is increased up to around 170 μmol L -1 for toluidine blue and 200 μmol L -1 for methylene blue. At higher concentrations, the aggregation process leads to structures with lower anisometry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanobiologic Research in a Microgravity Environment Bioreactor

NASA Astrophysics Data System (ADS)

Guidi, A.; Dubini, G.; Tominetti, F.; Raimondi, M.

A current problem in tissue culturing technology is the unavailability of an effective Bioreactor for the in vitro cultivation of cells and explants. It has, in fact, proved extremely difficult to promote the high-density three-dimensional in vitro growth of human tissues that have been removed from the body and deprived of their normal in vivo vascular sources of nutrients and gas exchange. A variety of tissue explants can be maintained for a short period of time on a supportive collagen matrix surrounded by culture medium. But this system provides only limited mass transfer of nutrients and wastes through the tissue, and gravity-induced sedimentation prevents complete three- dimensional cell-cell and cell-matrix interactions. Several devices presently on the market have been used with only limited success since each has limitations, which restrict usefulness and versatility. Further, no Bioreactor or culture vessel is known that will allow for unimpeded growth of three dimensional cellular aggregates or tissue. Extensive research on the effect of mechanical stimuli on cell metabolism suggests that tissues may respond to mechanical stimulation via loading-induced flow of the interstitial fluids. During the culture, cells are subject to a flow of culture medium. Flow properties such as flow field, flow regime (e.g. turbulent or laminar), flow pattern (e.g. circular), entity and distribution of the shear stress acting on the cells greatly influence fundamental aspects of cell function, such as regulation and gene expression. This has been demonstrated for endothelial cells and significant research efforts are underway to elucidate these mechanisms in various other biological systems. Local fluid dynamics is also responsible of the mass transfer of nutrients and catabolites as well as oxygenation through the tissue. Most of the attempts to culture tissue-engineered constructs in vitro have utilized either stationary cultures or systems generating relatively small mechanical forces. For example, cartilage constructs have been cultured in spinner flasks under mixed or unmixed conditions, in simulated and in real microgravity. In these mixing studies, however, it is difficult to definitively quantify the effects of mixing-induced mechanical forces from those of convection-enhanced transport of nutrients to and of catabolites away from the cells. At the state of the art, the presence of a more controlled mechanical environment may be the condition required in order to study the biochemical and mechanical response of these biological systems. Such a controlled environment could lead to an advanced fluid dynamic design of the culture chamber that could both enhance the local mass transfer phenomena and match the needs of specific macroscopic mechanical effects in tissue development. The bioreactor is an excellent example of how the skills and resources of two distinctly different fields can complement each other. Microgravity can be used to enhance the formation of tissue like aggregates in specially designed bioreactors. Theoretical and experimental projects are under way to improve cell culture techniques using microgravity conditions experienced during space flights. Bioreactors usable under space flight conditions impose constructional principles which are different from those intended solely for ground applications. The Columbus Laboratory as part of the International Space Station (ISS) will be an evolving facility in low Earth orbit. Its mission is to support scientific, technological, and commercial activities in space. A goal of this research is to design a unique bioreactor for use sequentially from ground research to space research. One of the particularities of the simulated microgravity obtained through time averaging of the weight vector is that by varying the rotational velocity the same results can be obtained with a different value of g. One of the first applications of this technique in space biology was in fact the Rotating Wall Vessel developed by NASA, and originally designed to protect cell culture from the high shear forces generated during the launch and the landing of the Space Shuttle. A Bioreactor that is used both for ground and flight experiments provides the additional benefit of isolating dependent variable of gravity. This continuity will provide a means to compare results to a control experiment.

Three dimensional optic tissue culture and process

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor); Goodwin, Thomas J. (Inventor); Francis, Karen M. (Inventor); Cardwell, Delmar R. (Inventor); Oconnor, Kim (Inventor); Fitzgerald, Wendy S. (Inventor); Aten, Laurie A. (Inventor)

1994-01-01

A process for artificially producing three-dimensional optic tissue has been developed. The optic cells are cultured in a bioreactor at low shear conditions. The tissue forms normal, functional tissue organization and extracellular matrix.

Fabrication of three-dimensional scaffolds using precision extrusion deposition with an assisted cooling device.

PubMed

Hamid, Q; Snyder, J; Wang, C; Timmer, M; Hammer, J; Guceri, S; Sun, W

2011-09-01

In the field of biofabrication, tissue engineering and regenerative medicine, there are many methodologies to fabricate a building block (scaffold) which is unique to the target tissue or organ that facilitates cell growth, attachment, proliferation and/or differentiation. Currently, there are many techniques that fabricate three-dimensional scaffolds; however, there are advantages, limitations and specific tissue focuses of each fabrication technique. The focus of this initiative is to utilize an existing technique and expand the library of biomaterials which can be utilized to fabricate three-dimensional scaffolds rather than focusing on a new fabrication technique. An expanded library of biomaterials will enable the precision extrusion deposition (PED) device to construct three-dimensional scaffolds with enhanced biological, chemical and mechanical cues that will benefit tissue generation. Computer-aided motion and extrusion drive the PED to precisely fabricate micro-scaled scaffolds with biologically inspired, porosity, interconnectivity and internal and external architectures. The high printing resolution, precision and controllability of the PED allow for closer mimicry of tissues and organs. The PED expands its library of biopolymers by introducing an assisting cooling (AC) device which increases the working extrusion temperature from 120 to 250 °C. This paper investigates the PED with the integrated AC's capabilities to fabricate three-dimensional scaffolds that support cell growth, attachment and proliferation. Studies carried out in this paper utilized a biopolymer whose melting point is established to be 200 °C. This polymer was selected to illustrate the newly developed device's ability to fabricate three-dimensional scaffolds from a new library of biopolymers. Three-dimensional scaffolds fabricated with the integrated AC device should illustrate structural integrity and ability to support cell attachment and proliferation.

CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

PubMed

Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

2015-02-01

Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data indicate that CD147 and CD98 might play important roles in cyclophilin-induced cell migration. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Complex structures from patterned cell sheets

PubMed Central

Misra, M.; Audoly, B.; Shvartsman, S. Y.

2017-01-01

The formation of three-dimensional structures from patterned epithelial sheets plays a key role in tissue morphogenesis. An important class of morphogenetic mechanisms relies on the spatio-temporal control of apical cell contractility, which can result in the localized bending of cell sheets and in-plane cell rearrangements. We have recently proposed a modified vertex model that can be used to systematically explore the connection between the two-dimensional patterns of cell properties and the emerging three-dimensional structures. Here we review the proposed modelling framework and illustrate it through the computational analysis of the vertex model that captures the salient features of the formation of the dorsal appendages during Drosophila oogenesis. This article is part of the themed issue ‘Systems morphodynamics: understanding the development of tissue hardware’. PMID:28348251

Engineering three-dimensional cell mechanical microenvironment with hydrogels.

PubMed

Huang, Guoyou; Wang, Lin; Wang, Shuqi; Han, Yulong; Wu, Jinhui; Zhang, Qiancheng; Xu, Feng; Lu, Tian Jian

2012-12-01

Cell mechanical microenvironment (CMM) significantly affects cell behaviors such as spreading, migration, proliferation and differentiation. However, most studies on cell response to mechanical stimulation are based on two-dimensional (2D) planar substrates, which cannot mimic native three-dimensional (3D) CMM. Accumulating evidence has shown that there is a significant difference in cell behavior in 2D and 3D microenvironments. Among the materials used for engineering 3D CMM, hydrogels have gained increasing attention due to their tunable properties (e.g. chemical and mechanical properties). In this paper, we provide an overview of recent advances in engineering hydrogel-based 3D CMM. Effects of mechanical cues (e.g. hydrogel stiffness and externally induced stress/strain in hydrogels) on cell behaviors are described. A variety of approaches to load mechanical stimuli in 3D hydrogel-based constructs are also discussed.

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation.

PubMed

Huang, Hayden; Dong, Chen Y; Kwon, Hyuk-Sang; Sutin, Jason D; Kamm, Roger D; So, Peter T C

2002-04-01

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.

Chemical processing of three-dimensional graphene networks on transparent conducting electrodes for depleted-heterojunction quantum dot solar cells.

PubMed

Tavakoli, Mohammad Mahdi; Simchi, Abdolreza; Fan, Zhiyong; Aashuri, Hossein

2016-01-07

We present a novel chemical procedure to prepare three-dimensional graphene networks (3DGNs) as a transparent conductive film to enhance the photovoltaic performance of PbS quantum-dot (QD) solar cells. It is shown that 3DGN electrodes enhance electron extraction, yielding a 30% improvement in performance compared with the conventional device.

Example MODIS Global Cloud Optical and Microphysical Properties: Comparisons between Terra and Aqua

NASA Technical Reports Server (NTRS)

Hubanks, P. A.; Platnick, S.; King, M. D.; Ackerman, S. A.; Frey, R. A.

2003-01-01

MODIS observations from the NASA EOS Terra spacecraft (launched in December 1999, 1030 local time equatorial crossing) have provided a unique data set of Earth observations. With the launch of the NASA Aqua spacecraft in May 2002 (1330 local time), two MODIS daytime (sunlit) and nighttime observations are now available in a 24 hour period, allowing for some measure of diurnal variability. We report on an initial analysis of several operational global (Level-3) cloud products from the two platforms. The MODIS atmosphere Level-3 products, which include clear-sky and aerosol products in addition to cloud products, are available as three separate files providing daily, eight-day, and monthly aggregations; each temporal aggregation is spatially aggregated to a 1 degree grid. The files contain approximately 600 statisitical datasets (from simple means and standard deviations to 1 - and 2-dimensional histograms). Operational cloud products include detection (cloud fraction), cloud-top properties, and daytimeonly cloud optical thickness and particle effective radius for both water and ice clouds. We will compare example global Terra and Aqua cloud fraction, optical thickness, and effective radius aggregations.

AFM study of Escherichia coli RNA polymerase σ⁷⁰ subunit aggregation.

PubMed

Dubrovin, Evgeniy V; Koroleva, Olga N; Khodak, Yulia A; Kuzmina, Natalia V; Yaminsky, Igor V; Drutsa, Valeriy L

2012-01-01

The self-assembly of Escherichia coli RNA polymerase σ⁷⁰ subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ⁷⁰ subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ⁷⁰ polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ⁷⁰ subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ⁷⁰ subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ⁷⁰( subunit was investigated using atomic force microscopy and other complementary approaches. 2012 Elsevier Inc. All rights reserved.

Comparative study of aerogels obtained from differently prepared nanocellulose fibers.

PubMed

Chen, Wenshuai; Li, Qing; Wang, Youcheng; Yi, Xin; Zeng, Jie; Yu, Haipeng; Liu, Yixing; Li, Jian

2014-01-01

This article describes the fabrication of nanocellulose fibers (NCFs) with different morphologies and surface properties from biomass resources as well as their self-aggregation into lightweight aerogels. By carefully modulating the nanofibrillation process, four types of NCFs could be readily fabricated, including long aggregated nanofiber bundles, long individualized nanofibers with surface C6 -carboxylate groups, short aggregated nanofibers, and short individualized nanofibers with surface sulfate groups. Free-standing lightweight aerogels were obtained from the corresponding aqueous NCF suspensions through freeze-drying. The structure of the aerogels could be controlled by manipulating the type of NCFs and the concentration of their suspensions. A possible mechanism for the self-aggregation of NCFs into two- or three-dimensional aerogel nanostructures was further proposed. Owing to web-like structure, high porosity, and high surface reactivity, the NCF aerogels exhibited high mechanical flexibility and ductility, and excellent properties for water uptake, removal of dye pollutants, and the use as thermal insulation materials. The aerogels also displayed sound-adsorption capability at high frequencies. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proper Generalized Decomposition (PGD) for the numerical simulation of polycrystalline aggregates under cyclic loading

NASA Astrophysics Data System (ADS)

Nasri, Mohamed Aziz; Robert, Camille; Ammar, Amine; El Arem, Saber; Morel, Franck

2018-02-01

The numerical modelling of the behaviour of materials at the microstructural scale has been greatly developed over the last two decades. Unfortunately, conventional resolution methods cannot simulate polycrystalline aggregates beyond tens of loading cycles, and they do not remain quantitative due to the plasticity behaviour. This work presents the development of a numerical solver for the resolution of the Finite Element modelling of polycrystalline aggregates subjected to cyclic mechanical loading. The method is based on two concepts. The first one consists in maintaining a constant stiffness matrix. The second uses a time/space model reduction method. In order to analyse the applicability and the performance of the use of a space-time separated representation, the simulations are carried out on a three-dimensional polycrystalline aggregate under cyclic loading. Different numbers of elements per grain and two time increments per cycle are investigated. The results show a significant CPU time saving while maintaining good precision. Moreover, increasing the number of elements and the number of time increments per cycle, the model reduction method is faster than the standard solver.

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

PubMed

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

2016-10-01

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

Two-dimensional infrared correlation spectroscopy study of the aggregation of cytochrome c in the presence of dimyristoylphosphatidylglycerol.

PubMed Central

Paquet, M J; Laviolette, M; Pézolet, M; Auger, M

2001-01-01

Two-dimensional infrared correlation spectroscopy (2D-IR) was used in this study to investigate the aggregation of cytochrome c in the presence of dimyristoylphosphatidylglycerol. The influence of temperature on the aggregation has been evaluated by monitoring the intensity of a band at 1616 cm(-1), which is characteristic of aggregated proteins, and the 2D-IR analysis has been used to determine the various secondary structure components of cytochrome c involved before and during its aggregation. The 2D-IR correlation analysis clearly reveals for the first time that aggregation starts to occur between nearly native proteins, which then unfold, yielding to further aggregation of the protein. Later in the aggregation process, the formation of intermolecular bonds and unfolding of the alpha-helices appear to be simultaneous. These results lead us to propose a two-step aggregation process. Finally, the results obtained during the heating period clearly indicate that before the protein starts to aggregate, there is a loosening of the tertiary structure of cytochrome c, resulting in a decrease of the beta-sheet content and an increase of the amount of beta-turns. This study clearly demonstrates the potential of 2D-IR spectroscopy to investigate the aggregation of proteins and this technique could therefore be applied to other proteins such as those involved in fibrilogenesis. PMID:11423415

Three-dimensional radiation transfer modeling in a dicotyledon leaf

NASA Astrophysics Data System (ADS)

Govaerts, Yves M.; Jacquemoud, Stéphane; Verstraete, Michel M.; Ustin, Susan L.

1996-11-01

The propagation of light in a typical dicotyledon leaf is investigated with a new Monte Carlo ray-tracing model. The three-dimensional internal cellular structure of the various leaf tissues, including the epidermis, the palisade parenchyma, and the spongy mesophyll, is explicitly described. Cells of different tissues are assigned appropriate morphologies and contain realistic amounts of water and chlorophyll. Each cell constituent is characterized by an index of refraction and an absorption coefficient. The objective of this study is to investigate how the internal three-dimensional structure of the tissues and the optical properties of cell constituents control the reflectance and transmittance of the leaf. Model results compare favorably with laboratory observations. The influence of the roughness of the epidermis on the reflection and absorption of light is investigated, and simulation results confirm that convex cells in the epidermis focus light on the palisade parenchyma and increase the absorption of radiation.

Micropatterned cell-cell interactions enable functional encapsulation of primary hepatocytes in hydrogel microtissues.

PubMed

Li, Cheri Y; Stevens, Kelly R; Schwartz, Robert E; Alejandro, Brian S; Huang, Joanne H; Bhatia, Sangeeta N

2014-08-01

Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell-cell interactions and cell-matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell-cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug-drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms.

Influence of serum percentage on the behavior of Wharton's jelly mesenchymal stem cells in culture.

PubMed

Harmouch, C; El-Omar, R; Labrude, P; Decot, V; Menu, P; Kerdjoudj, H

2013-01-01

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several lineages with valuable applications in regenerative medicine. MSCs differentiation is highly dependent on physicochemical properties of the culture substrate, cell density and on culture medium composition. In this study, we assessed the influence of fetal bovine serum (FBS) level on Wharton's jelly (WJ)-MSCs behavior seeded on polyelectrolyte multilayer films (PEMF) made of four bilayers of poly-allylamine hydrochloride (PAH) as polycation and poly-styrene sulfonate (PSS) as polyanion. MSCs isolated from WJ by explants method were amplified until the third passage. Their phenotypic characterization was performed by flow cytometry analyses. MSCs were seeded on PEMF, in Endothelial growth medium-2 (EGM-2) supplemented by either 5% or 2% FBS. Cell's behavior was monitored for 20 days by optical microscopy and immunofluorescence. Until 2 weeks on glass slides, no difference was observed whatever the FBS percentage. Then with 5% FBS, MSCs formed three-dimensional spheroids on PSS/PAH after 20 days of culture with a nuclear aggregate. Whereas, with 2% FBS, these spheroids did not appear and cells grown in 2D conserved the fibroblast-like morphology. The decrease of FBS percentage from 5% to 2% avoids 3D cell spheroids formation on PAH/PSS. Such results could guide bioengineering towards building 2D structures like cell layers or 3D structures by increasing the osteogenic or chondrogenic differentiation potential of MSCs.

TgrC1 mediates cell-cell adhesion by interacting with TgrB1 via mutual IPT/TIG domains during development of Dictyostelium discoideum.

PubMed

Chen, Gong; Wang, Jun; Xu, Xiaoqun; Wu, Xiangfu; Piao, Ruihan; Siu, Chi-Hung

2013-06-01

Cell-cell adhesion plays crucial roles in cell differentiation and morphogenesis during development of Dictyostelium discoideum. The heterophilic adhesion protein TgrC1 (Tgr is transmembrane, IPT, IG, E-set, repeat protein) is expressed during cell aggregation, and disruption of the tgrC1 gene results in the arrest of development at the loose aggregate stage. We have used far-Western blotting coupled with MS to identify TgrB1 as the heterophilic binding partner of TgrC1. Co-immunoprecipitation and pull-down studies showed that TgrB1 and TgrC1 are capable of binding with each other in solution. TgrB1 and TgrC1 are encoded by a pair of adjacent genes which share a common promoter. Both TgrB1 and TgrC1 are type I transmembrane proteins, which contain three extracellular IPT/TIG (immunoglobulin, plexin, transcription factor-like/transcription factor immunoglobulin) domains. Antibodies raised against TgrB1 inhibit cell reassociation at the post-aggregation stage of development and block fruiting body formation. Ectopic expression of TgrB1 and TgrC1 driven by the actin15 promoter leads to heterotypic cell aggregation of vegetative cells. Using recombinant proteins that cover different portions of TgrB1 and TgrC1 in binding assays, we have mapped the cell-binding regions in these two proteins to Lys(537)-Ala(783) in TgrB1 and Ile(336)-Val(360) in TgrC1, corresponding to their respective TIG3 and TIG2 domain.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death*

PubMed Central

Itakura, Masanori; Kubo, Takeya; Kaneshige, Akihiro; Harada, Naoki; Izawa, Takeshi; Azuma, Yasu-Taka; Kuwamura, Mitsuru; Yamaji, Ryouichi; Takeuchi, Tadayoshi

2017-01-01

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death. PMID:28167533

Toward three-dimensional microelectronic systems: directed self-assembly of silicon microcubes via DNA surface functionalization.

PubMed

Lämmerhardt, Nico; Merzsch, Stephan; Ledig, Johannes; Bora, Achyut; Waag, Andreas; Tornow, Marc; Mischnick, Petra

2013-07-02

The huge and intelligent processing power of three-dimensional (3D) biological "processors" like the human brain with clock speeds of only 0.1 kHz is an extremely fascinating property, which is based on a massively parallel interconnect strategy. Artificial silicon microprocessors are 7 orders of magnitude faster. Nevertheless, they do not show any indication of intelligent processing power, mostly due to their very limited interconnectivity. Massively parallel interconnectivity can only be realized in three dimensions. Three-dimensional artificial processors would therefore be at the root of fabricating artificially intelligent systems. A first step in this direction would be the self-assembly of silicon based building blocks into 3D structures. We report on the self-assembly of such building blocks by molecular recognition, and on the electrical characterization of the formed assemblies. First, planar silicon substrates were functionalized with self-assembling monolayers of 3-aminopropyltrimethoxysilane for coupling of oligonucleotides (single stranded DNA) with glutaric aldehyde. The oligonucleotide immobilization was confirmed and quantified by hybridization with fluorescence-labeled complementary oligonucleotides. After the individual processing steps, the samples were analyzed by contact angle measurements, ellipsometry, atomic force microscopy, and fluorescence microscopy. Patterned DNA-functionalized layers were fabricated by microcontact printing (μCP) and photolithography. Silicon microcubes of 3 μm edge length as model objects for first 3D self-assembly experiments were fabricated out of silicon-on-insulator (SOI) wafers by a combination of reactive ion etching (RIE) and selective wet etching. The microcubes were then surface-functionalized using the same protocol as on planar substrates, and their self-assembly was demonstrated both on patterned silicon surfaces (88% correctly placed cubes), and to cube aggregates by complementary DNA functionalization and hybridization. The yield of formed aggregates was found to be about 44%, with a relative fraction of dimers of some 30%. Finally, the electrical properties of the formed dimers were characterized using probe tips inside a scanning electron microscope.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

NASA Astrophysics Data System (ADS)

Zhang, Haifeng; Mao, Xiyuan; Du, Zijing; Jiang, Wenbo; Han, Xiuguo; Zhao, Danyang; Han, Dong; Li, Qingfeng

2016-01-01

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model.

PubMed

Zhang, Haifeng; Mao, Xiyuan; Du, Zijing; Jiang, Wenbo; Han, Xiuguo; Zhao, Danyang; Han, Dong; Li, Qingfeng

2016-01-01

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo . The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

PubMed

Lankoff, Anna; Sandberg, Wiggo J; Wegierek-Ciuk, Aneta; Lisowska, Halina; Refsnes, Magne; Sartowska, Bożena; Schwarze, Per E; Meczynska-Wielgosz, Sylwia; Wojewodzka, Maria; Kruszewski, Marcin

2012-02-05

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Crystal structures of the diastereomeric salt pair of the prostaglandin intermediate 1 R, 2 S(+)- cis-2-hydroxycyclopent-4-enylacetic acid with S- and R- 1-phenylethylamine

NASA Astrophysics Data System (ADS)

Czugler, Mátyás; Csöregh, Ingeborg; Kálmán, Alajos; Faigl, Ferenc; Ács, Mária

1989-05-01

Crystal structures of an enantiomeric salt pair formed between 1 R,2 S- cis-2-hydroxycyclopent-4-enylacetic acid ( S-HCA) and R(+)-1-phenylethylamine ( R-PEA) and the corresponding S-PEA salt have been determined by X-ray crystallography. The S-HCA: R-PEA 1:1 salt ( R-HCA-PEA hereafter) is orthorhombic, P2 12 12 1, with the unit-cell parameters a = 5.806(1), b = 9.261(1), c = 27.624(2) Å and R = 0.056 for 1162 reflections at ambient temperature. The S-HCA: S-PEA 1:1 salt ( S-HCAPEA) is also orthorhombic, P2 12 12 1, with the unit-cell data a = 6.034(2), b = 11.840(7), c = 20.198(11) Å at 170 K, R = 0.082 for 1196 data measured at low temperatures (170 K). The R-HCAPEA salt has its two components assembled into an elongated rod-like shape via two-dimensional hydrogen bonding between cations and anions thus forming a well-ordered crystal. In contrast, the cation and anion in the S-HCAPEA salt forms a more globular aggregate and displays orientation disorder in the five-membered ring part of the anion and maintains an essentially one-dimensional hydrogen-bond network, while the total number of hydrogen bonds between cationic and anionic species remains three in both crystals.

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.

Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.

PubMed

Han, Yi; Liu, Xing-Mao; Liu, Hong; Li, Shi-Chong; Wu, Ben-Chuan; Ye, Ling-Ling; Wang, Qu-Wei; Chen, Zhao-Lie

2006-11-01

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

Live Cell Characterization of DNA Aggregation Delivered through Lipofection

PubMed Central

Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A.; Gratton, Enrico; Jones, Mark R

2015-01-01

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation. PMID:26013547

Full three-dimensional morphology evolution of amorphous thin films for atomic layer deposition

NASA Astrophysics Data System (ADS)

Jin, Lingpeng; Li, Yawei; Hu, Zhigao; Chu, Junhao

2018-04-01

We introduce a Monte Carlo model based on random deposition and diffusion limited aggregation in order to study the morphological evolution of deposition of nanofilm, which is difficult to carry out by the experimental methods. The instantaneous evolution of morphology and the corresponding parameters are observed when employing a novel perspective, modeling the aggregation of nanoscale units. Despite simplifying the chemical details, the simulation results qualitatively describe experiments with bulky precursors, and the strong dependence of growth rate on steric hindrance is obtained. Moreover, the well know behavior that the delay before steady growth is accurately predicted and analyzed based solely on modeling. Through this work, the great influence of steric hindrance on the initial stage of ALD is described.

Sub-nanometer surface chemistry and orbital hybridization in lanthanum-doped ceria nano-catalysts revealed by 3D electron microscopy.

PubMed

Collins, Sean M; Fernandez-Garcia, Susana; Calvino, José J; Midgley, Paul A

2017-07-14

Surface chemical composition, electronic structure, and bonding characteristics determine catalytic activity but are not resolved for individual catalyst particles by conventional spectroscopy. In particular, the nano-scale three-dimensional distribution of aliovalent lanthanide dopants in ceria catalysts and their effect on the surface electronic structure remains unclear. Here, we reveal the surface segregation of dopant cations and oxygen vacancies and observe bonding changes in lanthanum-doped ceria catalyst particle aggregates with sub-nanometer precision using a new model-based spectroscopic tomography approach. These findings refine our understanding of the spatially varying electronic structure and bonding in ceria-based nanoparticle aggregates with aliovalent cation concentrations and identify new strategies for advancing high efficiency doped ceria nano-catalysts.

Sculpting Cells with Play Doh.

ERIC Educational Resources Information Center

Way, Virginia A.

1982-01-01

Suggests using Play Doh to mold models of the nucleus, mitochondria, and inner cellular structures. Students can conceptualize the cell's structures as three-dimensional even though they appear two-dimensional under a microscope. Includes instructions for preparing homemade dough. (Author/JN)

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

PubMed Central

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

2016-01-01

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

Ultra-high aggregate bandwidth two-dimensional multiple-wavelength diode laser arrays

NASA Astrophysics Data System (ADS)

Chang-Hasnain, Connie

1994-04-01

Two-dimensional (2D) multi-wavelength vertical cavity surface emitting laser (VCSEL) arrays is promising for ultrahigh aggregate capacity optical networks. A 2D VCSEL array emitting 140 distinct wavelengths was reported by implementing a spatially graded layer in the VCSEL structure, which in turn creates a wavelength spread. In this program, we concentrated on novel epitaxial growth techniques to make reproducible and repeatable multi-wavelength VCSEL arrays.

Live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix.

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Comparison of Genotoxic Damage in Monolayer Cell Cultures and Three-Dimensional Tissue-Like Cell Assemblies

NASA Technical Reports Server (NTRS)

Behravesh, E.; Emami, K.; Wu, H.; Gonda, S.

2004-01-01

Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying I genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to iron charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50-70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1,100-bp LacI target as well as assessment of DNA,damage to the entire 45-kbp construct. Cultured cells were exposed to high-enerir on charged particles at Brookhaven National Laboratory s Alternating Gradient Synchrotron facility for a total dose of 0, 0.1, 0.25,0.5, 1.0, or 2.0 Gy and allowed to recover for 0, 1, or 7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the Lac1 target. However, no differences in mutational type were found between the 2D and the 3D samples. These 3D tissue-like model systems can reduce the uncertainty involved in extrapolating risk between in vitro cellular and in vivo models.

Competition between protein folding and aggregation: A three-dimensional lattice-model simulation

NASA Astrophysics Data System (ADS)

Bratko, D.; Blanch, H. W.

2001-01-01

Aggregation of protein molecules resulting in the loss of biological activity and the formation of insoluble deposits represents a serious problem for the biotechnology and pharmaceutical industries and in medicine. Considerable experimental and theoretical efforts are being made in order to improve our understanding of, and ability to control, the process. In the present work, we describe a Monte Carlo study of a multichain system of coarse-grained model proteins akin to lattice models developed for simulations of protein folding. The model is designed to examine the competition between intramolecular interactions leading to the native protein structure, and intermolecular association, resulting in the formation of aggregates of misfolded chains. Interactions between the segments are described by a variation of the Go potential [N. Go and H. Abe, Biopolymers 20, 1013 (1981)] that extends the recognition between attracting types of segments to pairs on distinct chains. For the particular model we adopt, the global free energy minimum of a pair of protein molecules corresponds to a dimer of native proteins. When three or more molecules interact, clusters of misfolded chains can be more stable than aggregates of native folds. A considerable fraction of native structure, however, is preserved in these cases. Rates of conformational changes rapidly decrease with the size of the protein cluster. Within the timescale accessible to computer simulations, the folding-aggregation balance is strongly affected by kinetic considerations. Both the native form and aggregates can persist in metastable states, even if conditions such as temperature or concentration favor a transition to an alternative form. Refolding yield can be affected by the presence of an additional polymer species mimicking the function of a molecular chaperone.

Multi-cellular, three-dimensional living mammalian tissue

NASA Technical Reports Server (NTRS)

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

1994-01-01

The present invention relates to a multicellular, three-dimensional, living mammalian tissue. The tissue is produced by a co-culture process wherein two distinct types of mammalian cells are co-cultured in a rotating bioreactor which is completely filled with culture media and cell attachment substrates. As the size of the tissue assemblies formed on the attachment substrates changes, the rotation of the bioreactor is adjusted accordingly.

Rolled-Up Optical and Electronic Components for On-Chip Integrative Applications

DTIC Science & Technology

2013-10-10

attracted broad interest to create new three- dimensional electronics such as wrapable solar cells , pressure sensors and paper displays. The adaption to...cone-like microtube cavities Rolled-up electronics 1. Energy storage elements based on hybrid organic/inorganic nanomembranes 2.High performance...fabricated in this way to detect and analyze individual cells , biomolecules, and their bioactivities. 3.2 Three-dimensional confinement in asymmetric

Quantitative volumetric Raman imaging of three dimensional cell cultures

NASA Astrophysics Data System (ADS)

Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

2017-03-01

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

Three-dimensional imaging of biological cells with picosecond ultrasonics

NASA Astrophysics Data System (ADS)

Danworaphong, Sorasak; Tomoda, Motonobu; Matsumoto, Yuki; Matsuda, Osamu; Ohashi, Toshiro; Watanabe, Hiromu; Nagayama, Masafumi; Gohara, Kazutoshi; Otsuka, Paul H.; Wright, Oliver B.

2015-04-01

We use picosecond ultrasonics to image animal cells in vitro—a bovine aortic endothelial cell and a mouse adipose cell—fixed to Ti-coated sapphire. Tightly focused ultrashort laser pulses generate and detect GHz acoustic pulses, allowing three-dimensional imaging (x, y, and t) of the ultrasonic propagation in the cells with ˜1 μm lateral and ˜150 nm depth resolutions. Time-frequency representations of the continuous-wavelet-transform amplitude of the optical reflectivity variations inside and outside the cells show GHz Brillouin oscillations, allowing the average sound velocities of the cells and their ultrasonic attenuation to be obtained as well as the average bulk moduli.

Liarozole inhibits transforming growth factor-β3–mediated extracellular matrix formation in human three-dimensional leiomyoma cultures

PubMed Central

Levy, Gary; Malik, Minnie; Britten, Joy; Gilden, Melissa; Segars, James; Catherino, William H.

2014-01-01

Objective To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. Design Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. Setting Laboratory study. Patient(s) None. Intervention(s) Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. Main Outcome Measure(s) Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. Result(s) Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. Conclusion(s) Liarozole decreased TGF-β3 and TGF-β3–mediated extracellular matrix expression in a 3D uterine leiomyoma culture system. PMID:24825427

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

PubMed

Lund, A W; Stegemann, J P; Plopper, G E

2009-01-01

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids.

PubMed

Murphy, Kaitlin C; Whitehead, Jacklyn; Zhou, Dejie; Ho, Steve S; Leach, J Kent

2017-12-01

Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE 2 ) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE 2 ). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE 2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE 2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE 2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE 2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE 2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs. Mesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers.

PubMed

Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

2014-05-01

Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur. © 2013.

Regulation of aggregate size and pattern by adenosine and caffeine in cellular slime molds

PubMed Central

2012-01-01

Background Multicellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold Dictyostelium discoideum, adenosine is known to increase the aggregate size and its antagonist caffeine reduces the aggregate size. However, it is not clear if the actions of adenosine and caffeine are evolutionarily conserved among other slime molds known to use structurally unrelated chemoattractants. We have examined how the known factors affecting aggregate size are modulated by adenosine and caffeine. Result Adenosine and caffeine induced the formation of large and small aggregates respectively, in evolutionarily distinct slime molds known to use diverse chemoattractants for their aggregation. Due to its genetic tractability, we chose D. discoideum to further investigate the factors affecting aggregate size. The changes in aggregate size are caused by the effect of the compounds on several parameters such as cell number and size, cell-cell adhesion, cAMP signal relay and cell counting mechanisms. While some of the effects of these two compounds are opposite to each other, interestingly, both compounds increase the intracellular glucose level and strengthen cell-cell adhesion. These compounds also inhibit the synthesis of cAMP phosphodiesterase (PdsA), weakening the relay of extracellular cAMP signal. Adenosine as well as caffeine rescue mutants impaired in stream formation (pde4- and pdiA-) and colony size (smlA- and ctnA-) and restore their parental aggregate size. Conclusion Adenosine increased the cell division timings thereby making large number of cells available for aggregation and also it marginally increased the cell size contributing to large aggregate size. Reduced cell division rates and decreased cell size in the presence of caffeine makes the aggregates smaller than controls. Both the compounds altered the speed of the chemotactic amoebae causing a variation in aggregate size. Our data strongly suggests that cytosolic glucose and extracellular cAMP levels are the other major determinants regulating aggregate size and pattern. Importantly, the aggregation process is conserved among different lineages of cellular slime molds despite using unrelated signalling molecules for aggregation. PMID:22269093

Regulation of aggregate size and pattern by adenosine and caffeine in cellular slime molds.

PubMed

Jaiswal, Pundrik; Soldati, Thierry; Thewes, Sascha; Baskar, Ramamurthy

2012-01-23

Multicellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold Dictyostelium discoideum, adenosine is known to increase the aggregate size and its antagonist caffeine reduces the aggregate size. However, it is not clear if the actions of adenosine and caffeine are evolutionarily conserved among other slime molds known to use structurally unrelated chemoattractants. We have examined how the known factors affecting aggregate size are modulated by adenosine and caffeine. Adenosine and caffeine induced the formation of large and small aggregates respectively, in evolutionarily distinct slime molds known to use diverse chemoattractants for their aggregation. Due to its genetic tractability, we chose D. discoideum to further investigate the factors affecting aggregate size. The changes in aggregate size are caused by the effect of the compounds on several parameters such as cell number and size, cell-cell adhesion, cAMP signal relay and cell counting mechanisms. While some of the effects of these two compounds are opposite to each other, interestingly, both compounds increase the intracellular glucose level and strengthen cell-cell adhesion. These compounds also inhibit the synthesis of cAMP phosphodiesterase (PdsA), weakening the relay of extracellular cAMP signal. Adenosine as well as caffeine rescue mutants impaired in stream formation (pde4- and pdiA-) and colony size (smlA- and ctnA-) and restore their parental aggregate size. Adenosine increased the cell division timings thereby making large number of cells available for aggregation and also it marginally increased the cell size contributing to large aggregate size. Reduced cell division rates and decreased cell size in the presence of caffeine makes the aggregates smaller than controls. Both the compounds altered the speed of the chemotactic amoebae causing a variation in aggregate size. Our data strongly suggests that cytosolic glucose and extracellular cAMP levels are the other major determinants regulating aggregate size and pattern. Importantly, the aggregation process is conserved among different lineages of cellular slime molds despite using unrelated signalling molecules for aggregation.

Biodynamic profiling of three-dimensional tissue growth techniques

NASA Astrophysics Data System (ADS)

Sun, Hao; Merrill, Dan; Turek, John; Nolte, David

2016-03-01

Three-dimensional tissue culture presents a more biologically relevant environment in which to perform drug development than conventional two-dimensional cell culture. However, obtaining high-content information from inside three dimensional tissue has presented an obstacle to rapid adoption of 3D tissue culture for pharmaceutical applications. Biodynamic imaging is a high-content three-dimensional optical imaging technology based on low-coherence interferometry and digital holography that uses intracellular dynamics as high-content image contrast. In this paper, we use biodynamic imaging to compare pharmaceutical responses to Taxol of three-dimensional multicellular spheroids grown by three different growth techniques: rotating bioreactor, hanging-drop and plate-grown spheroids. The three growth techniques have systematic variations among tissue cohesiveness and intracellular activity and consequently display different pharmacodynamics under identical drug dose conditions. The in vitro tissue cultures are also compared to ex vivo living biopsies. These results demonstrate that three-dimensional tissue cultures are not equivalent, and that drug-response studies must take into account the growth method.

Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation and Drug Therapeutics12

PubMed Central

Upreti, Meenakshi; Jamshidi-Parsian, Azemat; Koonce, Nathan A; Webber, Jessica S; Sharma, Sunil K; Asea, Alexzander AA; Mader, Mathew J; Griffin, Robert J

2011-01-01

Classic cancer research for several decades has focused on understanding the biology of tumor cells in vitro. However, extending these findings to in vivo settings has been impeded owing to limited insights on the impact of microenvironment on tumor cells. We hypothesized that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells, which exist in the tumor microenvironment. To that end, we have developed a system to grow three-dimensional cultures of GFP-4T1 mouse mammary tumor and 2H11 murine endothelial cells in hanging drops of medium in vitro. The presence of 2H11 endothelial cells in these three-dimensional cocultures was found to sensitize 4T1-GFP tumor cells to chemotherapy (Taxol) and, at the same time, protect cells from ionizing radiation. These spheroidal cultures can also be implanted into the dorsal skinfold window chamber of mice for fluorescence imaging of vascularization and disease progression/treatment response. We observed rapid neovascularization of the tumor-endothelial spheroids in comparison to tumor spheroids grown in nude mice. Molecular analysis revealed pronounced up-regulation of several proangiogenic factors in the tumor tissue derived from the tumor-endothelial spheroids compared with tumor-only spheroids. Furthermore, the rate of tumor growth from tumor-endothelial spheroids in mice was faster than the tumor cell-only spheroids, resulting in greater metastasis to the lung. This three-dimensional coculture model presents an improved way to investigate more pertinent aspects of the therapeutic potential for radiation and/or chemotherapy alone and in combination with antiangiogenic agents. PMID:22191001

Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation and Drug Therapeutics.

PubMed

Upreti, Meenakshi; Jamshidi-Parsian, Azemat; Koonce, Nathan A; Webber, Jessica S; Sharma, Sunil K; Asea, Alexzander Aa; Mader, Mathew J; Griffin, Robert J

2011-12-01

Classic cancer research for several decades has focused on understanding the biology of tumor cells in vitro. However, extending these findings to in vivo settings has been impeded owing to limited insights on the impact of microenvironment on tumor cells. We hypothesized that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells, which exist in the tumor microenvironment. To that end, we have developed a system to grow three-dimensional cultures of GFP-4T1 mouse mammary tumor and 2H11 murine endothelial cells in hanging drops of medium in vitro. The presence of 2H11 endothelial cells in these three-dimensional cocultures was found to sensitize 4T1-GFP tumor cells to chemotherapy (Taxol) and, at the same time, protect cells from ionizing radiation. These spheroidal cultures can also be implanted into the dorsal skinfold window chamber of mice for fluorescence imaging of vascularization and disease progression/treatment response. We observed rapid neovascularization of the tumor-endothelial spheroids in comparison to tumor spheroids grown in nude mice. Molecular analysis revealed pronounced up-regulation of several proangiogenic factors in the tumor tissue derived from the tumor-endothelial spheroids compared with tumor-only spheroids. Furthermore, the rate of tumor growth from tumor-endothelial spheroids in mice was faster than the tumor cell-only spheroids, resulting in greater metastasis to the lung. This three-dimensional coculture model presents an improved way to investigate more pertinent aspects of the therapeutic potential for radiation and/or chemotherapy alone and in combination with antiangiogenic agents.

Cholinesterases and cell proliferation in "nonstratified" and "stratified" cell aggregates from chicken retina and tectum.

PubMed

Vollmer, G; Layer, P G

1987-12-01

Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cell-to-cell relationships ("sorting out"), but these systems never form highly laminated aggregates ("nonstratified" R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly "stratified" aggregates ("RPE-aggregates", see Vollmer et al. 1984). The present comparative study of "stratified" and "nonstratified" aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates. During early aggregate formation, in both "stratified" and "nonstratified" aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2-3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE- and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Three Dimensional Optic Tissue Culture and Process

NASA Technical Reports Server (NTRS)

OConnor, Kim C. (Inventor); Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); Aten, Laurie A. (Inventor); Francis, Karen M. (Inventor); Caldwell, Delmar R. (Inventor); Prewett, Tacey L. (Inventor); Fitzgerald, Wendy S. (Inventor)

1999-01-01

A process for artificially producing three-dimensional optic tissue has been developed. The optic cells are cultured in a bioireactor at low shear conditions. The tissue forms as normal, functional tissue grows with tissue organization and extracellular matrix formation.

Three-Dimensional, Transgenic Cell Models to Quantify Space Genotoxic Effects

NASA Technical Reports Server (NTRS)

Gonda, S. R.; Sognier, M. A.; Wu, H.; Pingerelli, P. L.; Glickman, B. W.; Dawson, David L. (Technical Monitor)

1999-01-01

The space environment contains radiation and chemical agents known to be mutagenic and carcinogenic to humans. Additionally, microgravity is a complicating factor that may modify or synergize induced genotoxic effects. Most in vitro models fail to use human cells (making risk extrapolation to humans more difficult), overlook the dynamic effect of tissue intercellular interactions on genotoxic damage, and lack the sensitivity required to measure low-dose effects. Currently a need exists for a model test system that simulates cellular interactions present in tissue, and can be used to quantify genotoxic damage induced by low levels of radiation and chemicals, and extrapolate assessed risk to humans. A state-of-the-art, three-dimensional, multicellular tissue equivalent cell culture model will be presented. It consists of mammalian cells genetically engineered to contain multiple copies of defined target genes for genotoxic assessment,. NASA-designed bioreactors were used to coculture mammalian cells into spheroids, The cells used were human mammary epithelial cells (H184135) and Stratagene's (Austin, Texas) Big Blue(TM) Rat 2 lambda fibroblasts. The fibroblasts were genetically engineered to contain -a high-density target gene for mutagenesis (60 copies of lacl/LacZ per cell). Tissue equivalent spheroids were routinely produced by inoculation of 2 to 7 X 10(exp 5) fibroblasts with Cytodex 3 beads (150 micrometers in diameter). at a 20:1 cell:bead ratio, into 50-ml HARV bioreactors (Synthecon, Inc.). Fibroblasts were cultured for 5 days, an equivalent number of epithelial cells added, and the fibroblast/epithelial cell coculture continued for 21 days. Three-dimensional spheroids with diameters ranging from 400 to 600 micrometers were obtained. Histological and immunohistochemical Characterization revealed i) both cell types present in the spheroids, with fibroblasts located primarily in the center, surrounded by epithelial cells; ii) synthesis of extracellular matrix; and iii,, mitotic cells located throughout the spheroids. Spheroidal integrity and cell viability were retained for the 30-day test period after removal of spheroids from the bioreactor. Potential utility of this three-dimensional, transgenic model for genotoxicity was initially assessed by exposure of spheroids to 0-2 Gy neon at dose rates of 0.3 to 1.5 Gy/min (National Institute of Radiological Sciences, Chiba, Japan). Quantification of mutation at the lacl gene revealed a linear dose response for mutation induction. Limited sequencing analysis of mutant clones revealed higher frequencies of deletions and multiple base sequence changes with increasing dose. These results suggest that our three-dimensional, transgenic model is applicable to a wide variety of studies involving the quantification, identification, and characterization of genotoxicity incurred in space and on Earth. This model uniquely allows investigation of the interaction of relevant factors, namely cell-to-cell interactions and the mechanistic interaction of microgravity with radiation insults and DNA repair. Using this three-dimensional model will allow us to obtain dual genotoxic information (i.e., mutation rate plus chromosome aberration data) from the same system so that one endpoint can be used to reference the other, thereby increasing the fidelity of the data set. Moreover, the tissue-equivalent nature of the three-dimensional model provides high confidence for relevance of risk assessment, i.e., the establishment of quality factors directly applicable to the microgravity environment.

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds.

PubMed

Bracke, Marc E; Roman, Bart I; Stevens, Christian V; Mus, Liselot M; Parmar, Virinder S; De Wever, Olivier; Mareel, Marc M

2015-06-06

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Characteristics of tumor and host cells in 3-D simulated microgravity environment

NASA Astrophysics Data System (ADS)

Chopra, V.; Dinh, T.; Wood, T.; Pellis, N.; Hannigan, E.

Co-cultures of three-dimensional (3-D) constructs of one cell type with dispersed cells of a second cell type in low-shear rotating suspension cultures in simulated microgravity environment have been used to investigate invasive properties of normal and malignant cell types. We have shown that the epithelial and endothelial cells undergo a switch in characteristics when grown in an in vitro 3-D environment, that mimics the in vivo host environment as compared with conventional two-dimensional (2-D) monolayer cultures. Histological preparations and immunohistochemical staining procedures of cocultured harvests demonstrated various markers of interest: like collagen vimentin, mucin, elastin, fibrin, fibrinogen, cytokeratin, adhesion molecules and various angiogenic factors by tumor cells from gynecological cancer patients along with fibroblasts, endothelial cells and patient-derived mononuclear cells (n=8). The growth rate was enhanced 10-15 folds by 3-D cocultures of patient-derived cells as compared with 2-D monolayer cultures and 3-D monocultures. The production of interleukin-2, interleukin-6, interleukin -8, vascular endothelial cell growth factor, basic fibroblast growth factor, and angiogenin was studied by using ELISA and RT- PCR. Human umbilical vein-derived endothelial cell (HUVEC) were used to study the mitogenic response of the conditioned medium collected from 3-D monocultures and cocultures during proliferation and migration assays. The conditioned medium collected from 3-D cocultures of cancer cells also 1) increased the expression of message levels of vascular endothelial growth factor and its receptor flt-1 and KDR was observed by HUVEC, and 2) increased the expression of intracellular and vascular cell adhesion molecules on the surface of HUVEC, when measured by using Live cell ELISA assays and immunofluorescent staining as compared with 3-D monocultures of normal epithelial cells. There was an increase in production of 1) enzymatic activity that could generate bioactive angiostatin from purified human plasminogen, and 2) fibrin (red), mucin (blue), and elastic fiber (black) by cell aggregates of 3-D monocultures of patient-derived cells as compared with 3-D monocultures of normal epithelial cells. This coculture system can be used to study the effectiveness of various antiangiogenic agents on endothelial cell proliferation and migration and also the interaction of multiple cell types in a cost effective fashion, since it provides new insights into the invasive process and its effects on both invading and invaded cells.

Stem Cells in Aggregate Form to Enhance Chondrogenesis in Hydrogels

PubMed Central

Sridharan, BanuPriya; Lin, Staphany M.; Hwu, Alexander T.; Laflin, Amy D.; Detamore, Michael S.

2015-01-01

There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton’s jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance. PMID:26719986

High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

PubMed Central

Joshi, Pranav; Lee, Moo-Yeal

2015-01-01

High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology. PMID:26694477

Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease.

PubMed

Tripette, Julien; Alexy, Tamas; Hardy-Dessources, Marie-Dominique; Mougenel, Daniele; Beltan, Eric; Chalabi, Tawfik; Chout, Roger; Etienne-Julan, Maryse; Hue, Olivier; Meiselman, Herbert J; Connes, Philippe

2009-08-01

Recent evidence suggests that red blood cell aggregation and the ratio of hematocrit to blood viscosity (HVR), an index of the oxygen transport potential of blood, might considerably modulate blood flow dynamics in the microcirculation. It thus seems likely that these factors could play a role in sickle cell disease. We compared red blood cell aggregation characteristics, blood viscosity and HVR at different shear rates between sickle cell anemia and sickle cell hemoglobin C disease (SCC) patients, sickle cell trait carriers (AS) and control individuals (AA). Blood viscosity determined at high shear rate was lower in sickle cell anemia (n=21) than in AA (n=52), AS (n=33) or SCC (n=21), and was markedly increased in both SCC and AS. Despite differences in blood viscosity, both sickle cell anemia and SCC had similar low HVR values compared to both AA and AS. Sickle cell anemia (n=21) and SCC (n=19) subjects had a lower red blood cell aggregation index and longer time for red blood cell aggregates formation than AA (n=16) and AS (n=15), and a 2 to 3 fold greater shear rate required to disperse red blood cell aggregates. The low HVR levels found in sickle cell anemia and SCC indicates a comparable low oxygen transport potential of blood in both genotypes. Red blood cell aggregation properties are likely to be involved in the pathophysiology of sickle cell disease: the increased shear forces needed to disperse red blood cell aggregates may disturb blood flow, especially at the microcirculatory level, since red blood cell are only able to pass through narrow capillaries as single cells rather than as aggregates.

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Nikolov, Ivan; Skutella, Thomas; Just, Lothar

2007-01-01

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Fabrication of three-dimensional collagen scaffold using an inverse mould-leaching process.

PubMed

Ahn, SeungHyun; Lee, SuYeon; Cho, Youngseok; Chun, Wook; Kim, GeunHyung

2011-09-01

Natural biopolymers, such as collagen or chitosan, are considered ideal for biomedical scaffolds. However, low processability of the materials has hindered the fabrication of designed pore structures controlled by various solid freeform-fabrication methods. A new technique to fabricate a biomedical three-dimensional collagen scaffold, supplemented with a sacrificial poly(ethylene oxide) mould is proposed. The fabricated collagen scaffold shows a highly porous surface and a three-dimensional structure with high porosity as well as mechanically stable structure. To show its feasibility for biomedical applications, fibroblasts/keratinocytes were co-cultured on the scaffold, and the cell proliferation and cell migration of the scaffold was more favorable than that obtained with a spongy-type collagen scaffold.

McSnow: A Monte-Carlo Particle Model for Riming and Aggregation of Ice Particles in a Multidimensional Microphysical Phase Space

NASA Astrophysics Data System (ADS)

Brdar, S.; Seifert, A.

2018-01-01

We present a novel Monte-Carlo ice microphysics model, McSnow, to simulate the evolution of ice particles due to deposition, aggregation, riming, and sedimentation. The model is an application and extension of the super-droplet method of Shima et al. (2009) to the more complex problem of rimed ice particles and aggregates. For each individual super-particle, the ice mass, rime mass, rime volume, and the number of monomers are predicted establishing a four-dimensional particle-size distribution. The sensitivity of the model to various assumptions is discussed based on box model and one-dimensional simulations. We show that the Monte-Carlo method provides a feasible approach to tackle this high-dimensional problem. The largest uncertainty seems to be related to the treatment of the riming processes. This calls for additional field and laboratory measurements of partially rimed snowflakes.

Adult Lung Spheroid Cells Contain Progenitor Cells and Mediate Regeneration in Rodents With Bleomycin-Induced Pulmonary Fibrosis.

PubMed

Henry, Eric; Cores, Jhon; Hensley, M Taylor; Anthony, Shirena; Vandergriff, Adam; de Andrade, James B M; Allen, Tyler; Caranasos, Thomas G; Lobo, Leonard J; Cheng, Ke

2015-11-01

Lung diseases are devastating conditions and ranked as one of the top five causes of mortality worldwide according to the World Health Organization. Stem cell therapy is a promising strategy for lung regeneration. Previous animal and clinical studies have focused on the use of mesenchymal stem cells (from other parts of the body) for lung regenerative therapies. We report a rapid and robust method to generate therapeutic resident lung progenitors from adult lung tissues. Outgrowth cells from healthy lung tissue explants are self-aggregated into three-dimensional lung spheroids in a suspension culture. Without antigenic sorting, the lung spheroids recapitulate the stem cell niche and contain a natural mixture of lung stem cells and supporting cells. In vitro, lung spheroid cells can be expanded to a large quantity and can form alveoli-like structures and acquire mature lung epithelial phenotypes. In severe combined immunodeficiency mice with bleomycin-induced pulmonary fibrosis, intravenous injection of human lung spheroid cells inhibited apoptosis, fibrosis, and infiltration but promoted angiogenesis. In a syngeneic rat model of pulmonary fibrosis, lung spheroid cells outperformed adipose-derived mesenchymal stem cells in reducing fibrotic thickening and infiltration. Previously, lung spheroid cells (the spheroid model) had only been used to study lung cancer cells. Our data suggest that lung spheroids and lung spheroid cells from healthy lung tissues are excellent sources of regenerative lung cells for therapeutic lung regeneration. The results from the present study will lead to future human clinical trials using lung stem cell therapies to treat various incurable lung diseases, including pulmonary fibrosis. The data presented here also provide fundamental knowledge regarding how injected stem cells mediate lung repair in pulmonary fibrosis. ©AlphaMed Press.

Networking and rheology of concentrated clay suspensions "matured" in mineral medicinal water.

PubMed

Aguzzi, Carola; Sánchez-Espejo, Rita; Cerezo, Pilar; Machado, José; Bonferoni, Cristina; Rossi, Silvia; Salcedo, Inmaculada; Viseras, César

2013-09-10

This work studied the influence of "maturation" conditions (time and agitation) on aggregation states, gel structure and rheological behaviour of a special kind of pharmaceutical semisolid products made of concentrated clay suspensions in mineral medicinal water. Maturation of the samples was carried out in distilled and sulphated mineral medicinal water, both in static conditions (without agitation) and with manual stirring once a week, during a maximum period of three months. At the measured pH interval (7.5-8.0), three-dimensional band-type networks resulting from face/face contacts were predominant in the laminar (disc-like) clay suspensions, whereas the fibrous (rod-like) particles formed micro-aggregates by van der Waals attractions. The high concentration of solids in the studied systems greatly determined their behaviour. Rod-like sepiolite particles tend to align the major axis in aggregates promoted by low shearing maturation, whereas aggregates of disc-like smectite particles did not have a preferential orientation and their complete swelling required long maturation time, being independent of stirring. Maturation of both kinds of suspensions resulted in improved rheological properties. Laminar clay suspensions became more structured with time, independently from static or dynamic maturation conditions, whereas for fibrous clay periodic agitation was also required. Rheological properties of the studied systems have been related to aggregation states and networking mechanisms, depending on the type of clay minerals constituents. Physical stability of the suspensions was not impaired by the specific composition of the Graena medicinal water. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantitative analysis of three-dimensional biological cells using interferometric microscopy

NASA Astrophysics Data System (ADS)

Shaked, Natan T.; Wax, Adam

2011-06-01

Live biological cells are three-dimensional microscopic objects that constantly adjust their sizes, shapes and other biophysical features. Wide-field digital interferometry (WFDI) is a holographic technique that is able to record the complex wavefront of the light which has interacted with in-vitro cells in a single camera exposure, where no exogenous contrast agents are required. However, simple quasi-three-dimensional holographic visualization of the cell phase profiles need not be the end of the process. Quantitative analysis should permit extraction of numerical parameters which are useful for cytology or medical diagnosis. Using a transmission-mode setup, the phase profile represents the multiplication between the integral refractive index and the thickness of the sample. These coupled variables may not be distinct when acquiring the phase profiles of dynamic cells. Many morphological parameters which are useful for cell biologists are based on the cell thickness profile rather than on its phase profile. We first overview methods to decouple the cell thickness and its refractive index using the WFDI-based phase profile. Then, we present a whole-cell-imaging approach which is able to extract useful numerical parameters on the cells even in cases where decoupling of cell thickness and refractive index is not possible or desired.

Robustness of the Process of Nucleoid Exclusion of Protein Aggregates in Escherichia coli

PubMed Central

Neeli-Venkata, Ramakanth; Martikainen, Antti; Gupta, Abhishekh; Gonçalves, Nadia; Fonseca, Jose

2016-01-01

ABSTRACT Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. Combined with cell divisions, this generates heterogeneous aggregate distributions in subsequent cell generations. We studied the robustness of this process with differing medium richness and antibiotics stress, which affect nucleoid size, using multimodal, time-lapse microscopy of live cells expressing both a fluorescently tagged chaperone (IbpA), which identifies in vivo the location of aggregates, and HupA-mCherry, a fluorescent variant of a nucleoid-associated protein. We find that the relative sizes of the nucleoid's major and minor axes change widely, in a positively correlated fashion, with medium richness and antibiotic stress. The aggregate's distribution along the major cell axis also changes between conditions and in agreement with the nucleoid exclusion phenomenon. Consequently, the fraction of aggregates at the midcell region prior to cell division differs between conditions, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, from the location of the peak of anisotropy in the aggregate displacement distribution, the nucleoid relative size, and the spatiotemporal aggregate distribution, we find that the exclusion of detectable aggregates from midcell is most pronounced in cells with mid-sized nucleoids, which are most common under optimal conditions. We conclude that the aggregate management mechanisms of E. coli are significantly robust but are not immune to stresses due to the tangible effect that these have on nucleoid size. IMPORTANCE Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. From live single-cell microscopy studies of the robustness of this process to various stresses known to affect nucleoid size, we find that nucleoid size and aggregate preferential locations change concordantly between conditions. Also, the degree of influence of the nucleoid on aggregate positioning differs between conditions, causing aggregate numbers at midcell to differ in cell division events, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, we find that aggregate segregation to the cell poles is most pronounced in cells with mid-sized nucleoids. We conclude that the energy-free process of the midcell exclusion of aggregates partially loses effectiveness under stressful conditions. PMID:26728194

Size- and time-dependent growth properties of human induced pluripotent stem cells in the culture of single aggregate.

PubMed

Nath, Suman C; Horie, Masanobu; Nagamori, Eiji; Kino-Oka, Masahiro

2017-10-01

Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 10 1 -(2.8 ± 0.5) × 10 1 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 10 1 -(6.8 ± 1.1) × 10 2 cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 10 3 -(3.5 ± 1.1) × 10 3 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A dimensionally split Cartesian cut cell method for hyperbolic conservation laws

NASA Astrophysics Data System (ADS)

Gokhale, Nandan; Nikiforakis, Nikos; Klein, Rupert

2018-07-01

We present a dimensionally split method for solving hyperbolic conservation laws on Cartesian cut cell meshes. The approach combines local geometric and wave speed information to determine a novel stabilised cut cell flux, and we provide a full description of its three-dimensional implementation in the dimensionally split framework of Klein et al. [1]. The convergence and stability of the method are proved for the one-dimensional linear advection equation, while its multi-dimensional numerical performance is investigated through the computation of solutions to a number of test problems for the linear advection and Euler equations. When compared to the cut cell flux of Klein et al., it was found that the new flux alleviates the problem of oscillatory boundary solutions produced by the former at higher Courant numbers, and also enables the computation of more accurate solutions near stagnation points. Being dimensionally split, the method is simple to implement and extends readily to multiple dimensions.

Simplified method for the transverse bending analysis of twin celled concrete box girder bridges

NASA Astrophysics Data System (ADS)

Chithra, J.; Nagarajan, Praveen; S, Sajith A.

2018-03-01

Box girder bridges are one of the best options for bridges with span more than 25 m. For the study of these bridges, three-dimensional finite element analysis is the best suited method. However, performing three-dimensional analysis for routine design is difficult as well as time consuming. Also, software used for the three-dimensional analysis are very expensive. Hence designers resort to simplified analysis for predicting longitudinal and transverse bending moments. Among the many analytical methods used to find the transverse bending moments, SFA is the simplest and widely used in design offices. Results from simplified frame analysis can be used for the preliminary analysis of the concrete box girder bridges.From the review of literatures, it is found that majority of the work done using SFA is restricted to the analysis of single cell box girder bridges. Not much work has been done on the analysis multi-cell concrete box girder bridges. In this present study, a double cell concrete box girder bridge is chosen. The bridge is modelled using three- dimensional finite element software and the results are then compared with the simplified frame analysis. The study mainly focuses on establishing correction factors for transverse bending moment values obtained from SFA.

Composition and aggregation of extracellular polymeric substances (EPS) in hyperhaline and municipal wastewater treatment plants

PubMed Central

Zeng, Jie; Gao, Jun-Min; Chen, You-Peng; Yan, Peng; Dong, Yang; Shen, Yu; Guo, Jin-Song; Zeng, Ni; Zhang, Peng

2016-01-01

As important constituents of activated sludge flocs, extracellular polymeric substances (EPS) play significant roles in pollutants adsorption, the formation and maintenance of microbial aggregates, and the protection of microbes from external environmental stresses. In this work, EPS in activated sludge from a municipal wastewater treatment plant (M-WWTP) with anaerobic/anoxic/oxic (A2/O) process and a hyperhaline wastewater treatment plant (H-WWTP) with anaerobic/oxic (A/O) process were extracted by ultrasound method. The proteins and polysaccharides contents in EPS were determined by using a modified Lowry method and anthrone colorimetry respectively to analyze the detail differences in two types of WWTPs. Fourier transform-infrared spectroscopy and three-dimensional excitation-emission matrix fluorescence spectroscopy demonstrated proteins and polysaccharides were the dominant components of the two types of EPS, and the aromatic protein-like substances accounted for a larger proportion in EPS proteins. The results of the aggregation test indicated that EPS were good for the sludge aggregation, and the EPS in oxic sludge were more beneficial to sludge aggregation than that in anoxic sludge. Anoxic sludge EPS in H-WWTP showed a negligible effect on sludge aggregation. Comparative study on EPS of different tanks in the M-WWTP and H-WWTP was valuable for understanding the characteristics of EPS isolated from two typical wastewater treatment processes. PMID:27220287

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

PubMed

Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

2016-09-01

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Development and application of a three dimensional numerical model for predicting pollutant and sediment transport using an Eulerian-Lagrangian marker particle technique

NASA Technical Reports Server (NTRS)

Pavish, D. L.; Spaulding, M. L.

1977-01-01

A computer coded Lagrangian marker particle in Eulerian finite difference cell solution to the three dimensional incompressible mass transport equation, Water Advective Particle in Cell Technique, WAPIC, was developed, verified against analytic solutions, and subsequently applied in the prediction of long term transport of a suspended sediment cloud resulting from an instantaneous dredge spoil release. Numerical results from WAPIC were verified against analytic solutions to the three dimensional incompressible mass transport equation for turbulent diffusion and advection of Gaussian dye releases in unbounded uniform and uniformly sheared uni-directional flow, and for steady-uniform plug channel flow. WAPIC was utilized to simulate an analytic solution for non-equilibrium sediment dropout from an initially vertically uniform particle distribution in one dimensional turbulent channel flow.

Two-dimensional patterns in bacterial veils arise from self-generated, three-dimensional fluid flows.

PubMed

Cogan, N G; Wolgemuth, C W

2011-01-01

The behavior of collections of oceanic bacteria is controlled by metabolic (chemotaxis) and physical (fluid motion) processes. Some sulfur-oxidizing bacteria, such as Thiovulum majus, unite these two processes via a material interface produced by the bacteria and upon which the bacteria are transiently attached. This interface, termed a bacterial veil, is formed by exo-polymeric substances (EPS) produced by the bacteria. By adhering to the veil while continuing to rotate their flagella, the bacteria are able to exert force on the fluid surroundings. This behavior induces a fluid flow that, in turn, causes the bacteria to aggregate leading to the formation of a physical pattern in the veil. These striking patterns are very similar in flavor to the classic convection instability observed when a shallow fluid is heated from below. However, the physics are very different since the flow around the veil is mediated by the bacteria and affects the bacterial densities. In this study, we extend a model of a one-dimensional veil in a two-dimensional fluid to the more realistic two-dimensional veil in a three-dimensional fluid. The linear stability analysis indicates that the Peclet number serves as a bifurcation parameter, which is consistent with experimental observations. We also solve the nonlinear problem numerically and are able to obtain patterns that are similar to those observed in the experiments.

Multi-regime transport model for leaching behavior of heterogeneous porous materials.

PubMed

Sanchez, F; Massry, I W; Eighmy, T; Kosson, D S

2003-01-01

Utilization of secondary materials in civil engineering applications (e.g. as substitutes for natural aggregates or binder constituents) requires assessment of the physical and environment properties of the product. Environmental assessment often necessitates evaluation of the potential for constituent release through leaching. Currently most leaching models used to estimate long-term field performance assume that the species of concern is uniformly dispersed in a homogeneous porous material. However, waste materials are often comprised of distinct components such as coarse or fine aggregates in a cement concrete or waste encapsulated in a stabilized matrix. The specific objectives of the research presented here were to (1) develop a one-dimensional, multi-regime transport model (i.e. MRT model) to describe the release of species from heterogeneous porous materials and, (2) evaluate simple limit cases using the model for species when release is not dependent on pH. Two different idealized model systems were considered: (1) a porous material contaminated with the species of interest and containing inert aggregates and, (2) a porous material containing the contaminant of interest only in the aggregates. The effect of three factors on constituent release were examined: (1) volume fraction of material occupied by the aggregates compared to a homogeneous porous material, (2) aggregate size and, (3) differences in mass transfer rates between the binder and the aggregates. Simulation results confirmed that assuming homogeneous materials to evaluate the release of contaminants from porous waste materials may result in erroneous long-term field performance assessment.

Three dimensional amorphous silicon/microcrystalline silicon solar cells

DOEpatents

Kaschmitter, James L.

1996-01-01

Three dimensional deep contact amorphous silicon/microcrystalline silicon (a-Si/.mu.c-Si) solar cells which use deep (high aspect ratio) p and n contacts to create high electric fields within the carrier collection volume material of the cell. The deep contacts are fabricated using repetitive pulsed laser doping so as to create the high aspect p and n contacts. By the provision of the deep contacts which penetrate the electric field deep into the material where the high strength of the field can collect many of the carriers, thereby resulting in a high efficiency solar cell.

Three dimensional amorphous silicon/microcrystalline silicon solar cells

DOEpatents

Kaschmitter, J.L.

1996-07-23

Three dimensional deep contact amorphous silicon/microcrystalline silicon (a-Si/{micro}c-Si) solar cells are disclosed which use deep (high aspect ratio) p and n contacts to create high electric fields within the carrier collection volume material of the cell. The deep contacts are fabricated using repetitive pulsed laser doping so as to create the high aspect p and n contacts. By the provision of the deep contacts which penetrate the electric field deep into the material where the high strength of the field can collect many of the carriers, thereby resulting in a high efficiency solar cell. 4 figs.

Rolled-up Functionalized Nanomembranes as Three-Dimensional Cavities for Single Cell Studies

PubMed Central

2014-01-01

We use micropatterning and strain engineering to encapsulate single living mammalian cells into transparent tubular architectures consisting of three-dimensional (3D) rolled-up nanomembranes. By using optical microscopy, we demonstrate that these structures are suitable for the scrutiny of cellular dynamics within confined 3D-microenvironments. We show that spatial confinement of mitotic mammalian cells inside tubular architectures can perturb metaphase plate formation, delay mitotic progression, and cause chromosomal instability in both a transformed and nontransformed human cell line. These findings could provide important clues into how spatial constraints dictate cellular behavior and function. PMID:24598026

Annotated Gene and Proteome Data Support Recognition of Interconnections Between the Results of Different Experiments in Space Research

NASA Astrophysics Data System (ADS)

Bauer, Johann; Wehland, Markus; Pietsch, Jessica; Sickmann, Albert; Weber, Gerhard; Grimm, Daniela

2016-06-01

In a series of studies, human thyroid and endothelial cells exposed to real or simulated microgravity were analyzed in terms of changes in gene expression patterns or protein content. Due to the limitation of available cells in many space research experiments, comparative and control experiments had to be done in a serial manner. Therefore, detected genes or proteins were annotated with gene names and SwissProt numbers, in order to allow searches for interconnections between results obtained in different experiments by different methods. A crosscheck of several studies on the behavior of cytoskeletal genes and proteins suggested that clusters of cytoskeletal components change differently under the influence of microgravity and/or vibration in different cell types. The result that LOX and ISG15 gene expression were clearly altered during the Shenzhou-8 spaceflight mission could be estimated by comparison with the results of other experiments. The more than 100-fold down-regulation of LOX supports our hypothesis that the amount and stability of extracellular matrix have a great influence on the formation of three-dimensional aggregates under microgravity. The approximately 40-fold up-regulation of ISG15 cannot yet be explained in detail, but strongly suggests that ISGylation, an alternative form of posttranslational modification, plays a role in longterm cultures.

Exact solution of three-dimensional transport problems using one-dimensional models. [in semiconductor devices

NASA Technical Reports Server (NTRS)

Misiakos, K.; Lindholm, F. A.

1986-01-01

Several parameters of certain three-dimensional semiconductor devices including diodes, transistors, and solar cells can be determined without solving the actual boundary-value problem. The recombination current, transit time, and open-circuit voltage of planar diodes are emphasized here. The resulting analytical expressions enable determination of the surface recombination velocity of shallow planar diodes. The method involves introducing corresponding one-dimensional models having the same values of these parameters.

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines.

PubMed

Peel, D J; Johnson, S A; Milner, M J

1990-01-01

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.

Aneuploidy and asynchronous replication in non-alcholic fatty liver disease and cryptogenic cirrhosis.

PubMed

Laish, Ido; Mannasse-Green, Batya; Hadary, Ruth; Konikoff, Fred M; Amiel, Aliza; Kitay-Cohen, Yona

2016-11-15

Non-alcoholic fatty liver disease (NAFLD) and cryptogenic cirrhosis (CC), which is largely a late sequela of NAFLD, are considered pre-neoplastic conditions that might progress to hepatocellular carcinoma. Aneuploidy, telomere aggregates and synchronization of replication were evaluated as markers of genetic instability in these patients. Peripheral blood lymphocytes from 22 patients with NAFLD, 20 patients with CC and 20 age-matched healthy controls were analyzed. To determine random aneuploidy, we used the fluorescence in situ hybridization (FISH) with probes for chromosomes 9 and 18. The rate of aneuploidy was inferred from the fraction of cells revealing one, three or more hybridization signals per cell. Aggregate size was divided into three fusion groups of 2-5, 6-10 and 11-15 telomeres, relative to the size of a single telomere. The replication pattern was determined by FISH in two pairs of alleles, 15qter and 13qter. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. Significantly higher random aneuploidy rate was found in the CC patients than in the control group, and to a lesser degree in NAFLD patients. Telomere aggregates were insignificantly higher in both groups. Only patients with CC showed significantly higher rate of asynchronous replication with proportionately more cells with two single dots among the normal cells (p<0.001). These results likely reflect changes in gene replication and cell cycle progression in these conditions, possibly correlating with their malignant potential. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel chiral separation material: polymerized organogel formed by chiral gelators for the separation of D- and L-phenylalanine.

PubMed

Fu, Xinjian; Yang, Yang; Wang, Ningxia; Wang, Hong; Yang, Yajiang

2007-01-01

N-Stearine-N'-stearyl-L-phenylalanine, a chiral compound, was synthesized and used as a gelator for the gelation of polymerizable solvents, such as ss-hydroxyethyl methacrylate (HEMA), styrene, etc. The scanning electron microscope (SEM) images of the gelator aggregates show fibril-like helices, typical chiral aggregates with diameters of 100-200 nm. The solvent molecules were immobilized by capillary forces in the three-dimensional network structures of the organogels. The HEMA organogels containing crosslinker polyethylene glycol dimethacrylates (PEG200DMA) were subsequently polymerized by in situ UV irradiation. A porous polymerized organogels were obtained after removal of gelator aggregates through ethanol extraction. The chiral separation of D- and L-phenylalanine was carried out by the adsorption of the polymerized organogels. The adsorption efficiency of L-phenylalanine on the polymerized organogels was found to be dependent on the concentration of the gelator and crosslinker. (c) 2007 John Wiley & Sons, Ltd.

Cell Sheet-Based Tissue Engineering for Organizing Anisotropic Tissue Constructs Produced Using Microfabricated Thermoresponsive Substrates.

PubMed

Takahashi, Hironobu; Okano, Teruo

2015-11-18

In some native tissues, appropriate microstructures, including orientation of the cell/extracellular matrix, provide specific mechanical and biological functions. For example, skeletal muscle is made of oriented myofibers that is responsible for the mechanical function. Native artery and myocardial tissues are organized three-dimensionally by stacking sheet-like tissues of aligned cells. Therefore, to construct any kind of complex tissue, the microstructures of cells such as myotubes, smooth muscle cells, and cardiomyocytes also need to be organized three-dimensionally just as in the native tissues of the body. Cell sheet-based tissue engineering allows the production of scaffold-free engineered tissues through a layer-by-layer construction technique. Recently, using microfabricated thermoresponsive substrates, aligned cells are being harvested as single continuous cell sheets. The cell sheets act as anisotropic tissue units to build three-dimensional tissue constructs with the appropriate anisotropy. This cell sheet-based technology is straightforward and has the potential to engineer a wide variety of complex tissues. In addition, due to the scaffold-free cell-dense environment, the physical and biological cell-cell interactions of these cell sheet constructs exhibit unique cell behaviors. These advantages will provide important clues to enable the production of well-organized tissues that closely mimic the structure and function of native tissues, required for the future of tissue engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamics of an HIV-1 infection model with cell mediated immunity

NASA Astrophysics Data System (ADS)

Yu, Pei; Huang, Jianing; Jiang, Jiao

2014-10-01

In this paper, we study the dynamics of an improved mathematical model on HIV-1 virus with cell mediated immunity. This new 5-dimensional model is based on the combination of a basic 3-dimensional HIV-1 model and a 4-dimensional immunity response model, which more realistically describes dynamics between the uninfected cells, infected cells, virus, the CTL response cells and CTL effector cells. Our 5-dimensional model may be reduced to the 4-dimensional model by applying a quasi-steady state assumption on the variable of virus. However, it is shown in this paper that virus is necessary to be involved in the modeling, and that a quasi-steady state assumption should be applied carefully, which may miss some important dynamical behavior of the system. Detailed bifurcation analysis is given to show that the system has three equilibrium solutions, namely the infection-free equilibrium, the infectious equilibrium without CTL, and the infectious equilibrium with CTL, and a series of bifurcations including two transcritical bifurcations and one or two possible Hopf bifurcations occur from these three equilibria as the basic reproduction number is varied. The mathematical methods applied in this paper include characteristic equations, Routh-Hurwitz condition, fluctuation lemma, Lyapunov function and computation of normal forms. Numerical simulation is also presented to demonstrate the applicability of the theoretical predictions.

Fibroblasts regulate the migration of MCF7 mammary carcinoma cells in hydrated collagen gel.

PubMed

Rossi, L; Reverberi, D; Capurro, C; Aiello, C; Cipolla, M; Bonanno, M; Podestà, G

1994-01-01

We have defined a tissue culture method suitable to study cell-cell interactions in an environmental set close to in vivo conditions. It consists of heterotypic cell populations mixed together inside a collagen gel in a chamber slide for a period of up to 14 days. When the three-dimensional system is saturated, cells will start to move on the plastic surface as monolayers surrounding the gel, with a characteristic speed depending on cell type. Usually fibroblasts move fast, while epithelial cells demonstrate a much lower pace of migration. At any given time gel contraction can be measured, and thus the rate of cell expansion, by knowing the distance from the edge of the gel to the leading edge of cell migration. By using this approach it was found that MCF7 mammary carcinoma cells display a great variety of morphologies following their mixture with different fibroblastic cell lines. In particular, when MCF7 cells were mixed with fibroblasts from human fetus, dog thymus and rat kidney, they migrated up to the leading edge of the fibroblastic front as isolated single cells or as cellular aggregates, many of which became necrotic in time, or took on an elongated morphology. Selective necrosis of MCF7 cells was also induced with serum concentration of 15% and 20% FCS, but only when they were mixed with fibroblasts. No necrosis was induced in MCF7 cells cultured alone. From these observations it is suggested that necrosis may sometimes favor the detachment and infiltration of resistant epithelial tumor cells by increasing their autonomous behaviour. Fibroblasts seem to be instrumental in regulating this process.

Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.

PubMed

Walton, Katherine D; Kolterud, Asa

2014-09-04

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine(1). Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought(1). The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth(2). Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mechanical stretching for tissue engineering: two-dimensional and three-dimensional constructs.

PubMed

Riehl, Brandon D; Park, Jae-Hong; Kwon, Il Keun; Lim, Jung Yul

2012-08-01

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols.

Mechanical Stretching for Tissue Engineering: Two-Dimensional and Three-Dimensional Constructs

PubMed Central

Riehl, Brandon D.; Park, Jae-Hong; Kwon, Il Keun

2012-01-01

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols. PMID:22335794

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

PubMed

Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

ROCK and PRK-2 Mediate the Inhibitory Effect of Y-27632 on Polyglutamine Aggregation

PubMed Central

Shao, Jieya; Welch, William J.; Diamond, Marc I.

2009-01-01

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, H-1077, HA-1152), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal. PMID:18423405

Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

PubMed

Vazquez, Alexei

2013-01-01

The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM) corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM) is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM) neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

Preparation of a Three-Dimensional Full Thickness Skin Equivalent.

PubMed

Reuter, Christian; Walles, Heike; Groeber, Florian

2017-01-01

In vitro test systems are a promising alternative to animal models. Due to the use of human cells in a three-dimensional arrangement that allows cell-cell or cell-matrix interactions these models may be more predictive for the human situation compared to animal models or two-dimensional cell culture systems. Especially for dermatological research, skin models such as epidermal or full-thickness skin equivalents (FTSE) are used for different applications. Although epidermal models provide highly standardized conditions for risk assessment, FTSE facilitate a cellular crosstalk between the dermal and epidermal layer and thus can be used as more complex models for the investigation of processes such as wound healing, skin development, or infectious diseases. In this chapter, we describe the generation and culture of an FTSE, based on a collagen type I matrix and provide troubleshooting tips for commonly encountered technical problems.

Biofilm growth program and architecture revealed by single-cell live imaging

NASA Astrophysics Data System (ADS)

Yan, Jing; Sabass, Benedikt; Stone, Howard; Wingreen, Ned; Bassler, Bonnie

Biofilms are surface-associated bacterial communities. Little is known about biofilm structure at the level of individual cells. We image living, growing Vibrio cholerae biofilms from founder cells to ten thousand cells at single-cell resolution, and discover the forces underpinning the architectural evolution of the biofilm. Mutagenesis, matrix labeling, and simulations demonstrate that surface-adhesion-mediated compression causes V. cholerae biofilms to transition from a two-dimensional branched morphology to a dense, ordered three-dimensional cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture, and this growth pattern is controlled by a single gene. Competition analyses reveal the advantages of the dense growth mode in providing the biofilm with superior mechanical properties. We will further present continuum theory to model the three-dimensional growth of biofilms at the solid-liquid interface as well as solid-air interface.

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

PubMed

Knoch, Tobias A; Wachsmuth, Malte; Kepper, Nick; Lesnussa, Michael; Abuseiris, Anis; Ali Imam, A M; Kolovos, Petros; Zuin, Jessica; Kockx, Christel E M; Brouwer, Rutger W W; van de Werken, Harmen J G; van IJcken, Wilfred F J; Wendt, Kerstin S; Grosveld, Frank G

2016-01-01

The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture ( T2C ), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.

Chemotactic preferences govern competition and pattern formation in simulated two-strain microbial communities.

PubMed

Centler, Florian; Thullner, Martin

2015-01-01

Substrate competition is a common mode of microbial interaction in natural environments. While growth properties play an important and well-studied role in competition, we here focus on the influence of motility. In a simulated two-strain community populating a homogeneous two-dimensional environment, strains competed for a common substrate and only differed in their chemotactic preference, either responding more sensitively to a chemoattractant excreted by themselves or responding more sensitively to substrate. Starting from homogeneous distributions, three possible behaviors were observed depending on the competitors' chemotactic preferences: (i) distributions remained homogeneous, (ii) patterns formed but dissolved at a later time point, resulting in a shifted community composition, and (iii) patterns emerged and led to the extinction of one strain. When patterns formed, the more aggregating strain populated the core of microbial aggregates where starving conditions prevailed, while the less aggregating strain populated the more productive zones at the fringe or outside aggregates, leading to a competitive advantage of the less aggregating strain. The presence of a competitor was found to modulate a strain's behavior, either suppressing or promoting aggregate formation. This observation provides a potential mechanism by which an aggregated lifestyle might evolve even if it is initially disadvantageous. Adverse effects can be avoided as a competitor hinders aggregate formation by a strain which has just acquired this ability. The presented results highlight both, the importance of microbial motility for competition and pattern formation, and the importance of the temporal evolution, or history, of microbial communities when trying to explain an observed distribution.

Mechanics of the Cell

NASA Astrophysics Data System (ADS)

Boal, David

2012-01-01

Preface; List of symbols; 1. Introduction to the cell; 2. Soft materials and fluids; Part I. Rods and Ropes: 3. Polymers; 4. Complex filaments; 5. Two-dimensional networks; 6. Three-dimensional networks; Part II. Membranes: 7. Biomembranes; 8. Membrane undulations; 9. Intermembrane and electrostatic forces; Part III. The Whole Cell: 10. Structure of the simplest cells; 11. Dynamic filaments; 12. Growth and division; 13. Signals and switches; Appendixes; Glossary; References; Index.

Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation.

PubMed

Shen, Francis H; Werner, Brian C; Liang, Haixiang; Shang, Hulan; Yang, Ning; Li, Xudong; Shimer, Adam L; Balian, Gary; Katz, Adam J

2013-01-01

Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning. Multicellular aggregate cells expressed increased differentiation potential and extracellular matrix production over the same human ADS cells cultured in monolayer. Furthermore, MAs reseeded onto monolayer retained their stem cell capabilities. When implanted in vivo, significantly greater bone volume and extracellular matrix were present in the implanted specimens of MAs confirmed on both microCT and histological sectioning. This is the first study to investigate the capability of human ADS cells in a 3D culture system to undergo osteogenic differentiation. The results confirm that MAs maintain their stem cell characteristics. Compared with analogous cells in monolayer culture, the human ADS cells as MAs exhibit elevated levels of osteogenic differentiation and increased matrix mineralization. Furthermore, the creation of uniform spheroids allows for improved handling and manipulation during transplantation. These findings strongly support the concept that 3D culture systems remain not only a viable option for stem cell culture but also possibly a more attractive alternative to traditional culture techniques to improve the osteogenic potential of human adipose stem cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Atomic Force Microscopy Based Cell Shape Index

NASA Astrophysics Data System (ADS)

Adia-Nimuwa, Usienemfon; Mujdat Tiryaki, Volkan; Hartz, Steven; Xie, Kan; Ayres, Virginia

2013-03-01

Stellation is a measure of cell physiology and pathology for several cell groups including neural, liver and pancreatic cells. In the present work, we compare the results of a conventional two-dimensional shape index study of both atomic force microscopy (AFM) and fluorescent microscopy images with the results obtained using a new three-dimensional AFM-based shape index similar to sphericity index. The stellation of astrocytes is investigated on nanofibrillar scaffolds composed of electrospun polyamide nanofibers that has demonstrated promise for central nervous system (CNS) repair. Recent work by our group has given us the ability to clearly segment the cells from nanofibrillar scaffolds in AFM images. The clear-featured AFM images indicated that the astrocyte processes were longer than previously identified at 24h. It was furthermore shown that cell spreading could vary significantly as a function of environmental parameters, and that AFM images could record these variations. The new three-dimensional AFM-based shape index incorporates the new information: longer stellate processes and cell spreading. The support of NSF PHY-095776 is acknowledged.

Tuning calcium carbonate growth through physical confinement and templating with amyloid-like polypeptide aggregates

NASA Astrophysics Data System (ADS)

Colaco, Martin Francis

The creation of useful composite materials requires precise control of the interface between the components in order to tune the overall shape and material properties. Despite the current research into nanotechnology, our ability to create materials with nanoscale precision is nascent. However, nature has a paradigm for the creation of finely structured composites under mild conditions called biomineralization. Through control of protein template assembly, solution conditions, and physical confinement, organisms are able to create useful optical and structural materials, such as bones, teeth, and mollusk shells. The objective of this thesis is to elucidate the importance of these various controls in synthetic systems to further our ability to create nanostructured materials. We begin by examining the formation of self-assembled monolayers (SAMs) of organosilanes on silica oxides. The formation of functionalized surfaces can help control the mineralization of amorphous or crystalline calcium carbonate. Long-chained organosilanes organize on surfaces to form dense, solid-like films, with the terminal groups determining the hydrophobicity and stereochemistry of the film. Our work has shown that uniform hydrophobic and hydrophilic films can be formed by using cleaned silica over glass or mica and through a vapor phase reaction over a liquid one. Additionally, we showed that mixed SAMs with phase-separated domains could be created through the selection of organosilanes and reaction conditions. We have built on these functionalized surfaces through the use of microfabrication and a gas permeable polymer to create three-dimensionally confined microcrystallizers. Other researchers have shown that one-dimensional confinement with a multi-functional surface (patterned with a small nucleating ordered region in a disordered SAM) can stabilize the creation of an amorphous calcium carbonate film before a single, large, micropatterned crystal is grown. Our work has determined that this methodology does not extend to three-dimensional confined systems, as the water has no method of escape. Through the addition of an insoluble hydroscopic polymer to our microreactors, amorphous calcium carbonate of controllable sizes can be grown. However, crystalline calcium carbonate cannot be grown without some type of templating. Studies of calcium carbonate templating have predominantly been performed on SAMs or in poorly characterized gels or protein films. The use of ordered protein or polypeptide aggregates for templating permits both geometry and charge surface density to be varied. We have studied the kinetics and final morphology of ordered aggregates of poly-L-glutamic acid and a copolymer of glutamic acid and alanine through experiments and simulations. Electrostatics, not structure, of the monomer appeared to be the dominating factor in the aggregation, as pH and salt concentration changes led to dramatic changes in the kinetics. Examining our experimental with existing models provided inconsistent results, so we developed a new model that yielded physically realistic rate constants, while generating better fits with longer lag phases and faster growths. However, despite the similarity of aggregation conditions, the two polypeptides yielded vastly different morphologies, with the PEA forming typical amyloid-like fibrils and PE forming larger, twisted lamellar aggregates. Templating with these aggregates also yielded dramatically different patterns. Polycrystalline rhombohedral calcite with smooth faces and edges grew on PEA fibrils, with minimal templating in evidence. However, on PE, numerous calcite crystals with triangular projections tracked the surface of the aggregate. The PE lamellae are characterized by extensive beta-sheet structure. In this conformation, the glutamic acid spacings on the surface of the aggregates can mimic the spacings of the carboxylates in the calcite lattice. In addition, the high negative charge density on the polypeptide surface led to a large number of nucleation sites. As the crystals grow, they impinge on each other but are limited to grow in one direction, perpendicular to the aggregate surface. Thus, the crystal structure propagates even at large length scales.

Crystallization of calcium oxalate monohydrate at dipalmitoylphosphatidylcholine monolayers in the presence of chondroitin sulfate A

NASA Astrophysics Data System (ADS)

Ouyang, Jian-Ming; Deng, Sui-Ping; Zhong, Jiu-Ping; Tieke, Bernd; Yu, Shu-Hong

2004-10-01

The growth and aggregation of calcium oxalate monohydrate (COM) crystals beneath dipalmitoylphosphatidylcholine (DPPC) monolayers in the presence of chondroitin sulfate A (C4S) was systematically examined under different surface pressure. The results indicated that the addition of C4S can inhibit the crystal growth and prevent the aggregation of COM crystals. Under a DPPC monolayer, well-defined three-dimensional hexagonal prisms and three-dimensional rhombus prisms with sharply angled tips were obtained. The DPPC monolayer at a surface pressure of 10 mN/m can match the Ca2+ distance of the (1 bar 0 1) face of COM better than at 20 mN/m. The addition of C4S could cooperatively modulate the interaction strength between the monolayer (or itself) with the specific morphology determining faces such as (1 bar 0 1) and (0 2 0), and thus results in remarkable stabilization of the (1 bar 0 1) faces. The dramatic changes in morphological details were due to the strong electrostatic interactions between the Ca2+-rich (1 bar 0 1) crystal faces of COM and the polyanionic polysaccharide C4S together with the negatively charged sites of the zwitterionic DPPC monolayers. The increase of the concentration of C4S can further enhance the stabilization of the (1 bar 0 1) face.