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Laboratory process provides environmental conditions favoring simultaneous growth of cocultures of mammalian cells of more than one type. Cultures become three-dimensional tissuelike assemblies serving as organoid models of differentiation of cells. Process used, for example, to study growth of human colon cancers, starting from mixtures of normal colonic fibroblasts and partially differentiated colon adenocarcinoma cells.
The potential for differentiation and radiosensitivity of the stem hemopoietic cells (KOE) under conditions of initial and later hypokinesia is examined. It is established that in the initial period of hypokinesia (3 days) when a stress reaction prevails, changes occur in the erythroid differentiation and radiosensitivity of KOE. This effect is associated with redistribution of T-lymphocytes that increase in number in the bone marrow of mice during hypokinesia. At later periods of hypokinesia (30 days) when changes in the organism are related to hypokinesia proper, differentiation and radiosensitivity of KOE were normalized.
The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.
The im electroporated injection of a protease-resistant GH-releasing hormone cDNA into rat dams at 16 d gestation resulted in enhanced long-term growth of the F(1) offspring. The offspring were significantly heavier by 2 wk of age, and the difference was sustained to 10 wk of age. Consistent with their augmented growth, the plasma IGF-I concentration of the F(1) progeny was increased significantly. The pituitary gland of the offspring was significantly heavier and contained an increased number of somatotrophs and PRL-secreting cells, which is indicative of modification of cell lineage differentiation. These unique findings demonstrate that enhanced GH-releasing hormone expression in pregnant dams can result in intergenerational growth promotion by altering development of the pituitary gland in the offspring.
Cell and animal studies conducted onboard the International Space Station and during the Shuttle program have provided extensive data illustrating bone degenerative responses to mechanical unloading in microgravity. Specifically CDKN1a/p21, an inhibitory modulator of cell cycle progression, is upregulated in osteoprecursor cells of the femur during 15-day spaceflight, suggesting that microgravity can block stem cell-based tissue regenerative process at the level of progenitor proliferation and differentiation. To study a potential role for CDKN1a/p21 in regulating osteogenic mechanosensitivity, we cultured primary bone marrow osteoprogenitor cells from CDKN1a/p21-null (p21-null) and wildtype mice with and without mechanical stimulation, and compared their morphological, proliferative, and in-vitro mineralization responses. Structural cell alterations due to mechanical stimulation were assessed by florescence labeling of f-actin cytoskeleton and focal adhesions. Mechanical stimulation of p21-null cells resulted in more pronounced cytoskeletal alignment with the axis of stretch than for wildtype cells. In addition, p21-null cells subjected to stretch loading also formed significantly more focal adhesions than wildtype cells. Combined these findings suggest that p21-null cells are structurally more responsive to stretch stimulation than the wildtype cells. Because osteoprogenitor cells are well known to respond to mechanical stimulation with increased proliferation, we also tested this response in p21-null cells. Results from those experiments show the proliferative capacity of mechanically stimulated p21-null cells far exceeded that of wildtype controls. Specifically, cell counts from 14, and 21 days post mechanical stimulation, show that p21- null cells to have a 4-fold increase in proliferation compared to wildtype. When the p21-null cell differentiation response to mechanical stimulation was evaluated, the p21-null cultuers elicited more extensive mineralization at earlier assessed timepoints than control cultures. Specifically, Von Kossa staining for mineralized matrix showed that the p21-null cells produced more than twice the mineralized surface area of wildtype cells, and at an earlier 7-day time point in culture. Taken together these results suggest that CDKN1a/p21 normally plays a role in negatively regulating osteoprogenitor proliferation and differentiation responses to mechanostimulation in bone. Findings of CDKN1a/p21's increased expression during spaceflight in microgravity also suggest not only a potential molecular mechanism for arresting regenerative bone growth in space, but potentially also a reduced impact for bone-formation-promoting exercise mechanostimulation. The findings described here constitute a novel role for p21 as a regulator of tissue regeneration in response to mechanical load stimulation, and also suggest a new promising molecular target to promote regenerative health in disuse conditions.
An electrophoretic separation method was applied to the separation of cells. To determine the efficiency of the separation, it was necessary to apply existing methodology and develop new methods to assess the characteristics and functions of the separated subpopulations. Through appropriate application of the widely used isoelectric focusing procedure, a reproducible separation method was developed. Cells accumulated at defined pH and 70-80% remained viable. The cells were suitable for further biologic, biochemical and immunologic studies.
A pair of spool valves are described which are balanced between pressures of reactant gases supplied to a fuel cell power plant. The pressure differences are controlled between the gases so as to maintain those pressures substantially in the proportions necessary for operation of the fuel cell.
Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.
Spaceflight experiments since 1981 have demonstrated that certain cell functions are altered by micro-g. Biophysical models suggest that cell membranes and organelles should not be affected directly by gravity, however, the chemical microenvironment surrounding the cell and molecular transport could be altered by reduced gravity. Most experiments have used suspended live cells in small chambers without stirring or medium exchange. Flight results include increased attachment of anchorage-dependent human cells to collagen coated microcarriers, reduced secretion of growth hormone from pituitary cells, decreased mitogenic response of lymphocytes, increased Interferon-alpha by lymphocytes, increased Interleukin-1 and Tumor Necrosis Factor secretion by macrophages. Related experiments on cells immediately postflight and on procaryotic cells have shown significant changes in secretory capacity, cell proliferation, differentiation and development. Postulated mechanism include altered cell-cell interactions, altered calcium ion transport, effects on cell cytoskeleton, transport of transmitters and interactions with receptors. The discussion includes use of new molecular methods, considerations for cell environmental control and a preview of several experiments planned for the Shuttle and Spacelab flights to study the basic effects of microgravity on cellular physiology and potential interactions of spaceflight with radiation damage and cellular repair mechanisms.
Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that showed signs of damage by viruses and virus titers (see figure) that indicated large increases in the populations of viruses during the days following inoculation.
The work presented has focussed on increasing the efficiency of InP-based solar cells through the development of a high-performance InP/Ga(0.47)In(0.53)As two-junction, three-terminal monolithic tandem cell. Such a tandem is particularly suited to space applications where a radiation-hard top cell (i.e., InP) is required. Furthermore, the InP/Ga(0.47)In(0.53)As materials system is lattice matched and offers a top cell/bottom cell bandgap differential (0.60 eV at 300 K) suitable for high tandem cell efficiencies under AMO illumination. A three-terminal configuration was chosen since it allows for independent power collection from each subcell in the monolithic stack, thus minimizing the adverse impact of radiation damage on the overall tandem efficiency. Realistic computer modeling calculations predict an efficiency boost of 7 to 11 percent from the Ga(0.47)In(0.53)As bottom cell under AMO illumination (25 C) for concentration ratios in the 1 to 1000 range. Thus, practical AMO efficiencies of 25 to 32 percent appear possible with the InP/Ga(0.47)In(0.53)As tandem cell. Prototype n/p/n InP/Ga(0.47)In(0.53)As monolithic tandem cells were fabricated and tested successfully. Using an aperture to define the illuminated areas, efficiency measurements performed on a non-optimized device under standard global illumination conditions (25 C) with no antireflection coating (ARC) give 12.2 percent for the InP top cell and 3.2 percent for the Ga(0.47)In(0.53)As bottom cell, yielding an overall tandem efficiency of 15.4 percent. With an ARC, the tandem efficiency could reach approximately 22 percent global and approximately 20 percent AMO. Additional details regarding the performance of individual InP and Ga(0.47)In(0.53)As component cells, fabrication and operation of complete tandem cells and methods for improving the tandem cell performance, are also discussed.
The maintenance of healthy adult tissues in mammals requires a complex homeostasis of molecular, cellular, tissue, and metabolic processes which are fundamentally different from the development and aging processes that bookend life. Cellular homeostasis in the adult requires molecular maintenance and repair of non-dividing cells such as cardiomyocytes and neurons, but also stem cell-based tissue regeneration via direct replacement of cell loss, such as in the blood, immune system, bone, skin, liver, intestine, and other tissues. Because stem cell-based tissue regenerative health requires constant proliferation and differentiation of stem cell progenitors in the bone marrow, and other adult stem cell niches, it is uniquely sensitive to the stresses of spaceflight including exposure to space radiation and mechanical unloading in microgravity. A key central hypothesis in this field is that those spaceflight stress factors can have profound negative effects on long-term tissue regenerative health mediated by adult stem cells, and that unmitigated, they may lead to premature tissue aging and functional failure. Specifically, it is thought that mechanical unloading due to lack of weight-bearing in space reduces mitogenic mechanotransduction necessary to promote adult stem cell proliferation and differentiation, and that space radiation can also lead to activation of cell cycle arrest mechanisms, further reducing adult stem cell proliferation. These hypotheses are being tested in low earth orbit(LEO)using a variety of cellular and whole organism tissue model systems, suggesting that spaceflight consistently interferes with stem cell tissue regenerative processes such as in mammalian embryoid bodies, regenerating newt tails, and mouse bone marrow hematopoietic and osteoprogenitor cells. Furthermore, potential molecular mechanisms integrating both space radiation and mechanical disuse via oxidative stress and the cell cycle inhibitor Cdkn1aare now under study using single cell (scRNAseq) expressome analysis of bone marrow osteoprogenitors, both under stretch loading, and spaceflight conditions including in various mouse transgenic null backgrounds relevant to these mechanisms. Future work in the area of tissue regenerative health in space for the coming decade should seek to understand the responses the various tissue regenerative stem cell niches in humans and relevant model organisms, and how they respond to long-term exposure to the space environment. Special emphases of future work should be on how regenerative deficits in whole-organism stem cell niches may lead to tissue degeneration and premature aging, and on the long-term proliferation and differentiation of stem cell derived tissue organoid models in the deep space environment outside of LEO.
Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.
Approximation method solution of nonlinear differential equation for time variation of overpotential during galvanostatic charging in electrochemical cell
Weightlessness and simulated weightlessness inhibit the rate of periosteal bone formation in long bones. Formation of preosteoblasts is suppressed in periodontal ligament (PDL) of maxillary molars, which suggests a generalized block in osteoblast histogenesis. Growth in length of long bones is decreased by simulated weightlessness, but there are no reliable data on the influence of actual weightlessness on metaphyseal growth. The nuclear size assay for assessing relative numbers of osteoblast precursor cells was utilized in the primary spongiosa of growing long bones subjected to actual and simulated weightlessness. It is found that: (1) Actual weightlessness decreases total number of osteogenic cells and inhibits differentiation of osteoblast precursor cells, (2) Simulated weightlessness suppresses only osteoblast differentation; and (3) The nuclear morphometric assay is an effective means of assessing osteogenic activity in the growing metaphysis or long bones.
Nonactomyosin component differentiation in potassium chloride insoluble myofilaments in vertebrate smooth muscle cells
Progress in immunology continues to grow exponentially every year. New applications of this knowledge are being developed for a broad range of clinical conditions. Conversely, the study of primary and secondary immunodeficiencies is helping to elucidate the intricate mechanisms of the immune system. We have selected a few of the most significant contributions to the fields of basic and clinical immunology published between October 2001 and October 2002. Our choice of topics in basic immunology included the description of T-bet as a determinant factor for T(H)1 differentiation, the role of the activation-induced cytosine deaminase gene in B-cell development, the characterization of CD4(+)CD25(+) regulatory T cells, and the use of dynamic imaging to study MHC class II transport and T-cell and dendritic cell membrane interactions. Articles related to clinical immunology that were selected for review include the description of immunodeficiency caused by caspase 8 deficiency; a case series report on X-linked agammaglobulinemia; the mechanism of action, efficacy, and complications of intravenous immunoglobulin; mechanisms of autoimmunity diseases; and advances in HIV pathogenesis and vaccine development. We also reviewed two articles that explore the possible alterations of the immune system caused by spaceflights, a new field with increasing importance as human space expeditions become a reality in the 21st century.
Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a three dimensional level of organization that could provide a more suitable model to study postnatal muscle development.