Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

 Microgravity interferes with numerous lymphocyte functions (expression of cell surface molecules, locomotion, polyclonal and antigen-specific activation, and the protein kinase C activity in signal transduction). The latter suggests that gravity may also affect programmed cell death (PCD) in lymphocyte populations. To test this hypothesis, we investigated spontaneous, activation- and radiation-induced PCD in peripheral blood mononuclear cells exposed to modeled microgravity (MMG) using a rotating cell culture system. The results showed significant inhibition of radiation- and activation-induced apoptosis in MMG and provide insights into the potential mechanisms of this phenomenon.

 Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (μ-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34 cells were cultured for 4–6 d in rotating wall vessels for simulation of μ-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34 cells cultured in normal gravity (1-g) proliferated up to threefold by day 4–6, cells cultured in μ-g did not increase in number. As a possible explanation for this, cells cultured in simulated μ-g were found to exit G₀/G₁ phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial μ-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in μ-g and 1-g–cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of μ-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.

 Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 × g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 × g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 × g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.

 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.

LA7 rat mammary tumor cells stimulate the proliferation, in culture, of three normal epithelial cell types, namely mouse mammary, rat mammary, and mouse thymic cells. Gap-junctional communication between LA7 feeders and mouse mammary cells was demonstrated by microinjection of lucifer yellow, which traveled from LA7 to the surrounding mouse mammary cells. The amount of ³H-uridine exchange between feeder and recipient mouse mammary, rat mammary, and mouse thymus cells correlated with the growth rate induced by the feeders. Cells of the Madin Darby canine kidney (MDCK) line, which do not appreciably stimulate mouse mammary cell growth when used as feeder cells, also exchange little ³H-uridine with them. Expression of connexins Cx43, 32, and 26 was studied in all these cell lines and strains by immunocytochemistry. Mouse mammary cells expressed Cx26, and a few mouse thymic cells expressed Cx32. LA7, mouse mammary, mouse thymic, and rat mammary cells all expressed easily detectable amounts of the gap-junction protein Cx43, in contrast to MDCK cells, which expressed only a hint of the protein. These results suggest that gap junctions composed of Cx43 are those by which the normal epithelial cells communicate with the LA feeders. Thus, the ability of feeder cells to stimulate proliferation in recipients correlates with the expression of Cx43 in both members of the feeder/recipient pair and the capacity to form functional gap junctions between these cells.

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.