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.

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.

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.

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.

Culture of hybridoma and Friend leukemia virus transformed cells in microgravity. Spacelab IML-1 mission.

The behaviour of two mammalian cell lines was investigated in Biorack during the 1st Spacelab international microgravity laboratory flight (IML-1) in the ESA facility Biorack. The parameters determined were cell proliferation, biosynthesis of specific cell products, consumption of glucose, glutamine and production of ammonia and lactate respectively. Murine Friend leukemia virus-transformed cells (Friend cells) were induced to differentiate and express hemoglobin (Hg) genes upon induction with dimethylsulfoxide (DMSO). No change was observed in all metabolic parameters including the production of Hg and the number of Hg-positive cells. Electron microscopy analysis showed no difference in morphology, mean cell volume and mitotic index between the different cell samples. Murine hybridoma cells revealed an increase (+ 30-40%) of cell proliferation rate in microgravity, whereas the metabolic parameters, production of monoclonal antibodies included, were lower in the 0 g than in the 1 g controls. The results clearly show that not all mammalian cells undergo dramatic changes in microgravity and that the effects reported on human T lymphocytes represent a unique case.

Activation of microcarrier-attached lymphocytes in microgravity.

A technology has been developed to achieve optimal attachment of adhesion-independent lymphocytes to microcarrier beads. The activation of T-lymphocytes by concanavalin A was tested under microgravity conditions in an experiment carried out in space during the first Spacelab Life Science Mission. Activation, measured as the synthesis of deoxyribonucleic acid (DNA) and the production of interferon-gamma, more than doubled in attached lymphocytes in microgravity. The depression of the activation discovered in previous space experiments is due to an impairment not of the lymphocyte but of the macrophage function. The system described here may be useful for radiobiological investigations on the effect of high-energy particles and for testing the efficiency of the immune system in humans during the long-duration space flight planned in the future. The biotechnological significance of the increased lymphokine production in space remains to be assessed.

Effect of head-down tilt bedrest (10 days) on lymphocyte reactivity.

Immunological responses of six healthy males to 10 days of head-down tilt bedrest (HDT) were assessed. Lymphocyte responsiveness was severely reduced immediately before, during, and immediately after the HDT, even though the lymphocyte numbers did not change. By contrast, delayed-type hypersensitivity was not affected. No dramatic changes were found in WBC counts and lymphocyte subpopulations, with the only exception of natural killer (NK) cells which transiently decreased immediately after HDT. Plasma cortisol levels were elevated above normal immediately before and during the HDT. The data suggest that the mitogenic response of lymphocytes was affected by psychological and fluid shift stress. These results are compared with data obtained during and after spaceflight. We conclude that the stress of HDT induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.

Effect of a herbal yeast food supplement and long-distance running on immunological parameters.

The effect of a food supplement on immunological parameters of 16 long-distance runners was tested in a randomized, double-blind and placebo-controlled trial. The supplement comprised plasmolysed herbal yeast, malt, honey, and orange juice. No statistically significant differences between the two groups regarding the following variables were detected at three sessions at rest and immediately after a 21 km run: total and differential white blood cell counts, numbers of B- and T-cells and T-subpopulations, concanavalin-A-induced lymphocyte proliferation, serum levels of immunoglobulins, neopterin, IL-2 receptors, beta 2-microglobulin, complement factor b, c4 and c3c, and c1-inactivator. These findings suggest that the effects of the tested food supplement on these parameters are negligible with respect to improvements in the immunological status of long-distance runners. The changes observed immediately after the run had a transient character. In both groups, however, low lymphocyte counts, IgG subclass 2 levels and c1-inactivator levels were noted at rest, which indicate that the immune status of endurance athletes may be affected by training.

Effect of long-term physical exercise on lymphocyte reactivity: similarity to spaceflight reactions.

The response of critical immunological parameters in seven athletes to the sustained physical stress of marathon running was assessed. Variables analysed were the responsiveness of lymphocytes (measured as mitogenic response to concanavalin A), the numbers of lymphocytes, their subsets, and leukocyte numbers. In addition, blood levels of cortisol, epinephrine, and norepinephrine were determined. After the run, lymphocyte responsiveness was severely depressed to 1-70% of the resting values, even though the lymphocyte counts did not change. Leukocyte counts were elevated 2.8-fold. No dramatic changes were found within the lymphocyte subsets, although an increase in pan T-cells and the helper/inducer subset 2 d after the run was significant. In addition, the numbers of B-cells decreased significantly. No change was observed within the suppressor/cytotoxic subset. Cortisol increased 2.1-fold, epinephrine 3.2-fold and norepinephrine 2.7-fold. All these parameters returned to baseline values within 2 d. These data were compared with data obtained during and after spaceflight. We conclude that prolonged physical stress of marathon running induces changes in immunological responsiveness that are strikingly similar to those arising from the stress of spaceflight.

Gravitational effects on mammalian cells.

In this paper we present first the results of our most recent investigations on gravitational effects on the activation of human lymphocytes: by immunoenzymatic staining and by using concanavalin A (Con A) coated to red blood cells (RBC) we demonstrate that the increase of activation measured at 10xg is due to a simultaneous activation of T- and B-lymphocytes whereas at 1xg only T-cells are stimulated. Conversely, activation of T-cells by chemical modification of the membrane with sodium periodate is depressed at 10xg. Secondly, experiments performed in the centrifuge as well as in the clinostat with Friend, K-562, and hybridoma cells show that each cell line develops its own adaptation reaction to gravitational stress.