Cell sensitivity to gravity.

Cultures of human lymphocytes exposed in microgravity to the mitogen concanavalin A showed less than 3 percent of the activation of ground controls. This result supports the hypothesis, based on simulations at low g and experiments at high g, that microgravity depresses whereas high gravity enhances cell proliferation rates. The effects of gravity are particularly strong in cells undergoing differentiation.

Key gravity-sensitive signaling pathways drive T cell activation.

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity.

The effect of hypogravity and hypergravity on cells of the immune system.

This article reviews the gravity effects discovered in T lymphocytes and other cells of the immune system. The strong depression of mitogenic activation first observed in an experiment conducted in Spacelab 1 in 1983 triggered several other investigations in space and on the ground in the clinostat and in the centrifuge in the past 10 years. During this period, great progress was made in our knowledge of the complex mechanism of T cell activation as well as the technology to analyze the lymphokines produced during stimulation. Nevertheless, several aspects of the steps leading to activation are not yet clear. Studies in hypogravity and hypergravity may contribute to answering some of the questions. A recent investigation in the U.S. Spacelab SLS-1, based on a new technology in which leukocytes are attached to microcarrier beads, showed that the strong inhibition of activation in microgravity is due to a malfunction of monocytes acting as accessory cells. In fact, interleukin-1 production is nearly nil in resuspended monocytes, whereas T cell activation is doubled in attached cells. In hypergravity, but not at 1g, concanavalin A bound to erythrocytes activates B lymphocytes in addition to T cells. The activation of Jurkat cells is also severely impaired in space. These recent results have raised new questions that have to be answered in experiments to be conducted in space and on Earth in this decade. The experimental system, based on the mitogenic activation of T lymphocytes and accessory cells attached to microcarriers, offers an optimum model for studying basic biological mechanisms of the cell to assess the immunological fitness of humans in space and to test the feasibility of bioprocesses in space as well as on Earth.

Mitogenic signal transduction in T lymphocytes in microgravity.

The activation by concanavalin A Con A of human peripheral blood lymphocytes (PBLs) in the presence of monocytes as accessory cells was investigated in cultures exposed to microgravity conditions in Spacelab. Activation of T cells was measured as incorporation of [3H]thymidine into DNA, secretion of interleukin-2 (IL-2), and interferon-gamma, and expression of IL-2 receptors. Whereas, as discovered in earlier experiments, the activation of resuspended T cells is strongly inhibited, activation of cells attached to microcarrier beads is more than doubled in microgravity. The results suggest that the depression of the activation in resuspended cells may be attributed to a malfunction of monocytes acting as accessory cells. In fact, although the ultrastructure of resuspended monocytes is not altered in microgravity, the secretion of IL-1 is strongly inhibited. Our data suggest that (1) IL-2 is produced independently of IL-1, (2) IL-1 production is triggered only when monocytes (and lymphocytes?) adhere to microcarriers, (3) the expression of IL-2 receptors depends on IL-1, and (4) provided sufficient IL-1 is available, activation is enhanced in microgravity. Finally, cultures of resuspended PBLs and monocytes in microgravity constitute a complete and natural system in which monocytes are not operational. This may be useful for studies of the role of accessory cells and cell-cell interactions in T lymphocyte activation.

Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen-activated T lymphocytes.

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin-2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of interleukin-2 and of its receptor in concanavalin A-activated lymphocytes with the RT-PCR technology. Microgravity conditions were simulated in the fast rotating clinostat and in the random positioning machine. The latter is an instrument introduced recently to study gravitational effects on single cells. Our data clearly show that the expression of both IL-2 and IL-2Ralpha genes is significantly inhibited in simulated O X g. Thus full activation is prevented.

Bioprocessing in space: human cells attach to beads in microgravity.

Attachment to a substrate and survival of human embryonic kidney (HEK) cells have been tested in an incubator installed in the flight-deck of the Space Shuttle 'Challenger' during its eighth mission. HEK cells are producing the enzyme urokinase and are presently investigated as candidates for electrophoretic separation in an apparatus developed and manufactured by McDonnell Douglas. Attachment of HEK cells to a substrate is mandatory for survival and production of urokinase after electrophoretic separation. Analysis of the samples shows that cells adhere, spread and survive in microgravity (< 10(-3) x g) conditions as well as the ground controls at 1 x g. This result represents an important step towards further bioprocessing in space.

Cultivation of Saccharomyces cerevisiae in a bioreactor in microgravity.

Yeast cells were cultured for 8 d in a newly developed bioreactor during the Spacelab IML-2 mission. Two bioreactors, one stirred and one without stirring, were installed in the Biorack facility in space. Two control units were installed in the Biorack module at the Kennedy Space Center. Samples were drawn on mission day 3, 5, 6, 7 and 8 and preserved either by freezing or chemically fixed for post-flight analysis. The values of pH, pH regulation, temperature and redox potential were transmitted on-line to the ground station throughout the mission. The performance of the bioreactor was satisfactory except for a partial failure of the medium micropump. Despite the failure of the pump, the data support the following conclusions: There is a significant difference in the distribution of the bud scars between cells cultured at 0 x g and at 1 x g. The percentage of randomly distributed bud scars was significantly higher in the flight (17%) than in the ground control cells (5%). No remarkable differences were noted in the cell cycle, ultrastructure, cell proliferation, cell volume, ethanol production and glucose consumption.

Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space.

The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

Activation signals of T lymphocytes in microgravity.

Human peripheral blood lymphocytes and monocytes were activated with concanavalin A with or without exogenous recombinant interleukin 1 (IL-1) alone or IL-1 + interleukin 2 (IL-2) under microgravity conditions to test the hypothesis that lack of production of IL-1 by monocytes is the cause of the near total loss of activation observed earlier on several Spacelab flights. The 60 min failure of the on-board 1 x g reference centrifuge at the time of the addition of the activator renders the in-flight data at 1 x g unreliable. However, the data from a previous experiment on SLS-1 show that there is no difference between the results from the in-flight 1 x g centrifuge and 1 x g on ground. The comparison between the data of the cultures at 0 x g in space and of the synchronous control at 1 x g on ground show that exogenous IL-1 and IL-2 do not prevent the loss of activity (measured as the mitotic index) at 0 x g; production of interferon-gamma, however, is partially restored. In contrast to a previous experiment in space, the production of IL-1 is not inhibited.

Movements and interactions of leukocytes in microgravity.

The mitogenic activation of human lymphocytes resuspended in vitro is dramatically reduced in microgravity. As cell-cell contacts are one of the elements essential for activation, the behaviour of human leukocytes (mainly lymphocytes and monocytes as accessory cells) in the presence of the mitogen concanavalin A was studied in the centrifuge microscope NIZEMI at 0 x g. Aggregates (formed by intercellular bindings of membrane glycoproteins via the tetravalent alpha-glucoside ligand concanavalin A) were found at 0 x g as well as at 1 x g already 12 h after the addition of the mitogen. In general, the aggregates observed at 0 x g after an incubation time of 46 and 78 h were smaller than the corresponding aggregates in the ground control. The findings are of primary importance since they confirm the indirect evidence we had from earlier Spacelab experiments and demonstrate that cell-cell contacts are occurring also in microgravity. In addition, single cells in 0 x g show a significant higher locomotion velocity than the cells at 1 x g. The fact that the locomotion capability is not decreased during the 78-h incubation with concanavalin A provides further evidence that the cells are not proceeding through the cell cycle.

Development of a miniature bioreactor for continuous culture in a space laboratory.

A new type of miniature bioreactor for continuous culture of yeast cells in space laboratories has been developed. Silicon microtechnology has permitted the integration of numerous functions and systems in a volume of 87 x 63 x 63 mm3 and a weight of 610 g. The 100 ml of fresh medium can be delivered at variable flow rates to the cultivation chamber (volume 3 ml) by means of a micropump. The culture is agitated by a magnetic stirrer. Microsensors monitor pH, temperature and redox potential. The decrease of pH occurring during the cultivation of Saccharomyces cerevisiae is compensated electrochemically. A window allows the inspection of the culture status. Samples of up to 1 ml can be drawn through a silicone rubber septum. The data measured by the sensors are transmitted on-line to the ground station during operations in space. The bioreactor had to fulfil several requirements related to the safety regulation of the space agencies. In particular, new materials had to be selected and tested for their biocompatibility. The instrument has now passed all space and biological qualification tests and will be used in an experiment selected by ESA for the International Microgravity Laboratory-2 Mission in Spacelab in July 1994. This paper gives the results of the functional and biological tests and a detailed description of the instrument.

Preservation of viable biological samples for experiments in space laboratories.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Human immune cells as space travelers.

Experiments in space have shown that T lymphocyte function is altered in more than 50% of space crew members. There is strong evidence that such effect is due to stress rather than to weightlessness per se. However the health of astronauts was never threatened so far. Experiments in-vitro with cultures of human peripheral blood lymphocytes (not from astronauts) have shown that T cell function is dramatically reduced. Recent work with the random positioning machine, a new instrument to simulate conditions similar to microgravity, indicate that there are direct gravitational effects on the genetic expression of interleukin-2 and of its receptor in T lymphocytes.

Bioprocessing under microgravity--an introduction.

The main biological processes presently considered for applications in space laboratories are: (i) bioseparation, (ii) cell cultivation and (iii) cell electrofusion. All three technologies were discussed by experts in the field at the 1988-COSPAR Meeting in Helsinki.

Theories and models of the biology of the cell in space--an introduction.

The World Space Congress 1992 took place after two Spacelab flights with important biological payloads on board, the SLS-1 (June 1991) and IML-1 (January 1992) missions respectively. Interesting experiments were carried out in 1991 also on the Shuttle middeck and on the sounding rocket MASER 4. The highlights of the investigations on these missions together with the results of relevant ground-based research were presented at the symposium.

Reduced lymphocyte activation in space: role of cell-substratum interactions.

We investigated the effect of substratum adhesiveness on stimulated lymphocyte blastogenesis by reducing and blocking cell adhesion with poly (2-hydroxyethyl methacrylate) (poly-HEMA) in a simple on-ground system. Cells grown on medium-thick and thick poly-HEMA films were rounded in shape and displayed no signs of spreading. By contrast, on tissue culture plastic and very thin poly-HEMA films, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was near zero when the cells were grown on the least adhesive substratum. On uncoated plastic, activated lymphocytes subjected to high gravity (20g) exhibited an increased proliferation rate (40%) compared with 1g. By contrast, on poly-HEMA, high gravity did not improve lymphocyte responsiveness. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.

Signal transduction in T lymphocytes--a comparison of the data from space, the free fall machine and the random positioning machine.

In this paper we discuss the effect of microgravity on T cells and we present the data of studies with two new machines for 0 g simulations. Several experiments in space show that mitogenic T cell activation is lost at 0 g. Immunocytochemistry indicates that such effect is associated with changes of the cytoskeleton. Biochemical studies suggest that the lack of expression of the interleukin-2 receptor is one of the major causes of the loss of activity. In fact, interleukin-2 is the third signal required for full activation. In order to deepen our investigations we are now working with the free-fall machine, FFM, invented by D. Mesland, and with the random positioning machine, RPM, or three-dimensional clinostat, developed by T. Hoson. The FFM produces periods of free-fall lasting approximately 800 ms followed by bounces of 15-30 g lasting 45-60 ms. The RPM eliminates the effect of gravity by rotating biological specimen randomly around two orthogonal axes. While the FFM failed to reproduce the results obtained with T lymphocytes in space, the data from the RPM are in good agreement with those in real microgravity. In fact, the inhibition of the mitotic index in the RPM is 89% compared to static controls. The RPM (as the FFM) can carry markedly larger specimen than the fast rotating clinostat and thus allows to conduct comprehensive studies to select suitable biological objects for further investigations in space.

Mammalian cell cultivation in space.

Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 microliters. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.

Signal transduction in T lymphocytes in microgravity.

More than 120 experiments conducted in space in the last 15 years have shown that dramatic changes are occurring in several types of single cells during their exposure to microgravity. One focus of today's research on cells in space is on signal transduction, especially those steps involving the cytoskeleton and cell-cell interactions. Signal transduction is often altered in microgravity as well as in hypergravity. This leads to changes in cell proliferation, genetic expression and differentiation. Interesting examples are leukocytes, HeLa cells, epidermoid cells and osteoblastic cells. Signalling pathways were studied in T lymphocytes in microgravity by several investigators after the discovery that mitogenic activation in vitro is virtually nil at 0g. T cells are a good model to study signal transduction because three extracellular signals (mitogen, IL-1 and IL-2) are required for full activation, and two classical pathways (via proteins G and PKC) are activated within the cell. In addition, low molecular weight GTP-binding proteins (Ras and Rap) are interacting with the cytoskeleton. The data at 0g support the notion that the expression of IL-2 receptor is inhibited at 0g, while mitogen binding and the transmission of IL-1 by accessory cells occur normally. In addition, alterations of the cytoskeleton suggest that the interaction with Rap proteins is disturbed. Data obtained with phorbol esters indicate that the function of PKC is changed in microgravity. Similar conclusions are drawn from the results with epidermoid cells A431.