Graviperception of lentil seedling roots grown in space (Spacelab Dl Mission)

The growth and graviresponsiveness of roots were investigated in lentil seedlings (Lens culinaris L. cv. Verte du Puy) grown (1) in microgravity, (2) on a 1 g centrifuge in space, (3) in microgravity and then placed on the 1 g centrifuge for 3 h, (4) on the ground. Dry seeds were hydrated in space (except for the ground control) and incubated for 25 h at 22°C in darkness. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in a Biorack glove box. Root length was similar for seedlings grown in space and for the ground and the 1 g centrifuge controls. The direction of root growth in the microgravity sample deviated strongly from the initial orientation of the roots of the dry seeds. This deviation could be due to spontaneous curvatures similar to those observed on clinostats. When lentil seedlings were first grown in microgravity for 25 h and then placed on the 1 g centrifuge for 3 h, their roots bent strongly under the effect of the centrifugal acceleration. The amplitude of root curvature on the centrifuge was not significantly different from that observed on ground controls growing in the vertical position and placed in the horizontal position for 3 h. The gravisensitivity of statocytes differentiated in microgravity was similar to that of statocytes differentiated on earth. There were no qualitative differences in the ultrastructural features of the gravisensing cells in microgravity and in the 1 g centrifuge and ground controls. However, the distribution of statoliths in the gravisensing cells was different in microgravity: most of them were observed in the proximal part of these cells. Thus, these organelles were not distributed at random, which is in contradiction with results obtained with clinostats. The distal complex of endoplasmic reticulum in the statocytes was not in contact with the amyloplasts. Contact and pressure of amyloplasts on the tubules were not prerequisites for gravisensing. The results obtained are not in agreement with the hypothesis that the distal endoplasmic reticulum would be the transducer of the action of the statoliths.     

Polarity of statocytes in lentil seedling roots grown in space (Spacelab D1 Mission)

A morphometric analysis of root statocytes was performed on seedlings of lentil ( Lens culinaris L., cv. Verte du Puy) in order to determine the effects of microgravity on the polarity of these cells. Seedlings were grown: (1) on the ground, (2) in microgravity, (3) on a 1 g centrifuge in space, (4) first in microgravity and then placed on a 1 g centrifuge for 3 h. Dry seeds were hydrated in space (except for the ground control) for 25 h in darkness at 22°C in the Biorack facility developed by the European Space Agency. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in the Biorack glove box. The average shape of the statocytes and the location of endoplasmic reticulum, amyloplasts and nucleus in the cells were analysed in the four samples. By considering the cell shape, it appears that the morphology of the statocytes on the ground was different from that observed in the space samples. Cell polarity was similar in microgravity and in the centrifuged samples except for the distribution of the amyloplasts. These organelles were not distributed at random in near zero gravity, and they were more numerous in the proximal than in the distal half. Moreover, the statoliths were more voluminous in microgravity than in the centrifuged samples. The nucleus was closer to the cell center in the statocytes of roots grown in microgravity than in statocytes of roots grown in microgravity and then placed on the 1 g centrifuge for 3 h. It is hypothesized that the nucleus is attached to the cell periphery and that its location is dependent upon gravity.     

Identification of proteins involved in inhibition of spheroid formation under microgravity

Many types of cells transit in vitro from a two‐ to a three‐dimensional growth, when they are exposed to microgravity. The underlying mechanisms are not yet understood. Hence, we investigated the impact of microgravity on protein content and growth behavior. For this purpose, the human thyroid cancer cells FTC‐133 were seeded either in recently developed cell containers that can endure enhanced physical forces and perform media changes and cell harvesting automatically or in T‐25 culture flasks. All cells were cultured for five days at 1g. Afterwards, a part of the cell containers were flown to the International Space Station, while another part was kept on the ground. T‐25 flasks were mounted on and next to a Random Positioning Machine. The cells were cultured for 12 days under the various conditions, before they were fixed with RNAlater. All fixed cultures showed monolayers, but three‐dimensional aggregates were not detected. In a subsequent protein analysis, 180 proteins were identified by mass spectrometry. These proteins did not indicate significant differences between cells exposed to microgravity and their 1g controls. However, they suggest that an enhanced production of proteins related to the extracellular matrix could detain the cells from spheroid formation, while profilin‐1 is phosphorylated.     

Effect of microgravity on the cell cycle in the lentil root

Characteristics of the cell cycle in cortical regions (0–0.6 mm from the root-cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1-g centrifuge for 29 h, (2) on the 1-g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1-g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1-g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1-g centrifuge in space and of the sample grown first in 1-g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1-g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could be due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1-g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.     

Simulated microgravity impairs respiratory burst activity in human promyelocytic cells

Summary The concept of microgravity (free-fall) influencing cellular functions in nonadherent cells has not been a part of mainstream scientific thought. Utilizing rotating wall vessels (RWVs) to generate simulated microgravity conditions, we found that respiratory burst activity was significantly altered in nonadherent promyelocytic (HL-60) cells. Specifically, HL-60 cells in simulated microgravity for 6, 19, 42, 47, and 49 d had 3.8-fold fewer cells that were able to participate in respiratory burst activity than cells from 1×g cultures (P=0.0011, N=5). The quantity of respiratory burst products from the cells in simulated microgravity was also significantly reduced. The fold increase over controls in mean fluorescence intensities for oxidative products from cells in microgravity was 1.1±0.1 versus 1.8±0.3 for cells at 1 ×g (P=0.013, N=4). Furthermore, the kinetic response for phorbol ester-stimulated burst activity was affected by simulated microgravity. These results demonstrate that simulated microgravity alters an innate cellular function (burst activity). If respiratory burst activity is impaired by true microgravity, then recovery from infections during spaceflight could be delayed. Finally, RWVs provide an excellent model for investigating the mechanisms associated with microgravity-induced changes in nonadherent cells.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.     

Effect of Change in Spindle Structure on Proliferation Inhibition of Osteosarcoma Cells and Osteoblast under Simulated Microgravity during Incubation in Rotating Bioreactor

In order to study the effect of microgravity on the proliferation of mammalian osteosarcoma cells and osteoblasts, the changes in cell proliferation, spindle structure, expression of MAD2 or BUB1, and effect of MAD2 or BUB1 on the inhibition of cell proliferation is investigated by keeping mammalian osteosarcoma cells and osteoblasts under simulated microgravity in a rotating wall vessel (2D-RWVS) bioreactor. Experimental results indicate that the effect of microgravity on proliferation inhibition, incidence of multipolar spindles, and expression of MAD2 or BUB1 increases with the extension of treatment time. And multipolar cells enter mitosis after MAD2 or BUB1 is knocked down, which leads to the decrease in DNA content, and decrease the accumulation of cells within multipolar spindles. It can therefore be concluded that simulated microgravity can alter the structure of spindle microtubules, and stimulate the formation of multipolar spindles together with multicentrosomes, which causes the overexpression of SAC proteins to block the abnormal cells in metaphase, thereby inhibiting cell proliferation. By clarifying the relationship between cell proliferation inhibition, spindle structure and SAC changes under simulated microgravity, the molecular mechanism and morphology basis of proliferation inhibition induced by microgravity is revealed, which will give experiment and theoretical evidence for the mechanism of space bone loss and some other space medicine problems.     

Cultivation of fall armyworm ovary cells in simulated microgravity

Summary A methodology is presented to culture Fall Armyworm Ovary cells in simulated microgravity using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 × 10⁷ viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm² in shaker culture to under 0.5 dyne/cm² in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.     

Persistence of plant hormone levels in rice shoots grown under microgravity conditions in space: its relationship to maintenance of shoot growth

We investigated the effects of microgravity environment on growth and plant hormone levels in dark‐grown rice shoots cultivated in artificial 1 g and microgravity conditions on the International Space Station (ISS). Growth of microgravity‐grown shoots was comparable to that of 1 g‐grown shoots. Endogenous levels of indole‐3‐acetic acid (IAA) in shoots remained constant, while those of abscisic acid (ABA), jasmonic acid (JA), cytokinins (CKs) and gibberellins (GAs) decreased during the cultivation period under both conditions. The levels of auxin, ABA, JA, CKs and GAs in rice shoots grown under microgravity conditions were comparable to those under 1 g conditions. These results suggest microgravity environment in space had minimal impact on levels of these plant hormones in rice shoots, which may be the cause of the persistence of normal growth of shoots under microgravity conditions. Concerning ethylene, the expression level of a gene for 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase, the key enzyme in ethylene biosynthesis, was reduced under microgravity conditions, suggesting that microgravity may affect the ethylene production. Therefore, ethylene production may be responsive to alterations of the gravitational force.     

Rac1 drives intestinal stem cell proliferation and regeneration

Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology.     

Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling

Faster growth and differentiation of liver stem cells to hepatocyte is one of the key factors during liver regeneration. In recent years, simulated microgravity, a physical force has shown to differentially regulate the differentiation and proliferation of stem cells. In the present work, we studied the effect of simulated microgravity on differentiation and proliferation of liver stem cells. The cells were subjected to microgravity, which was simulated using indigenously fabricated 3D clinostat. Proliferation, apoptosis, immunofluorescence assays and Western blot analysis were carried out to study the effects of simulated microgravity on liver stem cells. Microgravity treatment for 2 h enhanced proliferation of stem cells by twofold without inducing apoptosis and compromising cell viability. Analysis of hepatocyte nuclear factor 4‐α (HNF4‐α) expression after 2 h of microgravity treatment revealed that microgravity alone can induce the differentiation of stem cells within 2–3 days. Probing bone morphogenic protein 4 (BMP4) and Notch1 in microgravity treated stem cells elaborated downregulation of Notch1 and upregulation of BMP4 after 2 days of incubation. Further, blocking BMP4 using dorsomorphin and chordin conditioned media from chordin plasmid transfected cells attenuated microgravity mediated differentiation of liver stem cells. In conclusion, microgravity interplays with BMP4/Notch1 signaling in stem cells thus inducing differentiation of stem cells to hepatocytes. Present findings can be implicated in clinical studies where microgravity activated stem cells can regenerate the liver efficiently after liver injury. J. Cell. Biochem. 112: 1898–1908, 2011. © 2011 Wiley‐Liss, Inc.     

Modification of growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls grown under microgravity conditions in space

We carried out a space experiment, denoted as Aniso Tubule, to examine the effects of microgravity on the growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls, using lines in which microtubules are visualized by labeling tubulin or microtubule‐associated proteins (MAPs) with green fluorescent protein (GFP). In all lines, GFP‐tubulin6 (TUB6)‐, basic proline‐rich protein1 (BPP1)‐GFP‐ and spira1‐like3 (SP1L3)‐GFP‐expressing using a constitutive promoter, and spiral2 (SPR2)‐GFP‐ and GFP‐65 kDa MAP‐1 (MAP65‐1)‐expressing using a native promoter, the length of hypocotyls grown under microgravity conditions in space was longer than that grown at 1 g conditions on the ground. In contrast, the diameter of hypocotyls grown under microgravity conditions was smaller than that of the hypocotyls grown at 1 g. The percentage of cells with transverse microtubules was increased under microgravity conditions, irrespective of the lines. Also, the average angle of the microtubules with respect to the transverse cell axis was decreased in hypocotyls grown under microgravity conditions. When GFP fluorescence was quantified in hypocotyls of GFP‐MAP65‐1 and SPR2‐GFP lines, microgravity increased the levels of MAP65‐1, which appears to be involved in the maintenance of transverse microtubule orientation. However, the levels of SPR2 under microgravity conditions were comparable to those at 1 g. These results suggest that the microgravity‐induced increase in the levels of MAP65‐1 is involved in increase in the transverse microtubules, which may lead to modification of growth anisotropy, thereby developing longer and thinner hypocotyls under microgravity conditions in space.     

乳酸脱氢酶基因调控对HEK-293细胞代谢和腺病毒生产的影响

减少乳酸积累一直是哺乳动物细胞生物技术产业的一个目标。体外培养动物细胞时,乳酸积累主要是2种代谢途径作用的综合结果:一方面,葡萄糖在乳酸脱氢酶A(lactate dehydrogenase A,LDHA)的作用下生成乳酸;另一方面,乳酸可通过乳酸脱氢酶B(LDHB)或乳酸脱氢酶C(LDHC)氧化为丙酮酸重新进入三羧酸循环。本研究综合评估了乳酸代谢关键基因调控对人胚胎肾细胞(human embryonic kidney 293 cells,HEK-293)细胞生长、代谢和人腺病毒(human adenovirus,HAdV)生产的影响,有效提高了HEK-293细胞的HAdV生产能力,并为哺乳动物细胞的乳酸代谢工程调控提供了理论基础。通过改造乳酸代谢关键调控基因(敲除ldha基因以及过表达ldhb和ldhc基因),有效改善了HEK-293细胞的物质和能量代谢效率,显著提高了HAdV的生产。与对照细胞相比,3个基因改造均能促进细胞生长,降低乳酸和氨的积累,明显增强细胞的物质和能量代谢效率,显著提高了HEK-293细胞的HAdV生产能力。ldhc基因过表达对HEK-293细胞的生长、代谢和HAdV生产调控最显著,最大细胞密度提高了约38.7%,乳酸对葡萄糖得率和氨对谷氨酰胺得率分别下降了33.8%和63.3%,HAdV滴度提高了至少16倍。此外,相比于对照细胞株,改造细胞株的腺苷三磷酸(adenosine triphosphate,ATP)生成速率、ATP/O_(2)比率、ATP与腺苷二磷酸(adenosine diphosphate,ADP)的比值以及还原型辅酶Ⅰ(nicotinamide adenine dinucleotide,NADH)含量均有不同程度的提高,能量代谢效率明显改善。     

Microgravity alters basal and insulin-mediated metabolic activity of normal and neoplastic cells

In this paper we report the behaviour of normal vascular smooth muscle cells and transformed breast cancer cells under normal versus simulated microgravity conditions by comparing cell proliferation, Glucose transport, Methionine uptake and protein synthesis. Modeled microgravity profoundly affects cell growth (especially in normal cells) and Glucose or Methionine metabolism (although to different extent in the two cell lines). Since both cells own responsive insulin receptors, the comparison was extended to insulin-stimulated versus unstimulated conditions. We report that the detected metabolic changes were strongly enhanced when the cells were simultaneously stimulated with insulin and subjected to modeled microgravity stress. Such observations may have important returns for human health in space; they deserve further attention.     

Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO‐K1 cells

Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO‐K1 cell culture was investigated. For this purpose, CHO‐K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP‐expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications.     

Real microgravity condition promoted regeneration capacity of induced pluripotent stem cells during the TZ‐1 space mission

Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells that gained self‐renewal and differentiation capacity similar to embryonic stem cells. Taking the precious opportunity of the TianZhou‐1 spacecraft mission, we studied the effect of space microgravity (µg) on the self‐renewal capacity of iPSCs. Murine iPSCs carrying pluripotency reporter Oct4‐GFP were used. The Oct4‐EGFP‐iPSCs clones were loaded into the bioreactor and exposed to μg in outer space for 14 days. The control experiment was performed in identical device but on the ground in earth gravity (1 g). iPSCs clones were compact and highly expressed Oct4 before launch. In μg condition, cells in iPSC clones spread out more rapidly than those in ground 1 g condition during the first 3 days after launch. However, in 1 g condition, as the cell density increases, the Oct4‐GFP signal dropped significantly during the following 3 days. Interestingly, in μg condition, iPSCs originated from the spread‐out clones during the first 3 days appeared to cluster together and reform colonies that activated strong Oct4 expression. On the other hand, iPSC clones in 1 g condition were not able to recover Oct4 expression after overgrown. Our study for the first time performed real‐time imaging on the proliferation process of iPSCs in space and found that in μg condition, cell behaviour appeared to be more dynamic than on the ground.     

Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells

OBJECTIVES: Microgravity is known to affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, a few controversial findings have recently been reported with respect to the effects of microgravity on BMSC proliferation. Thus, we investigated the effects of simulated microgravity on rat BMSC (rBMSC) proliferation and their osteogeneic potential. MATERIALS AND METHODS: rBMSCs isolated from marrow using our established effective method, based on erythrocyte lysis, were identified by their surface markers and their proliferation characteristics under normal conditions. Then, they were cultured in a clinostat to simulate microgravity, with or without growth factors, and in osteogenic medium. Subsequently, proliferation and cell cycle parameters were assessed using methylene blue staining and flow cytometry, respectively; gene expression was determined using Western blotting and microarray analysis. RESULTS: Simulated microgravity inhibited population growth of the rBMSCs, cells being arrested in the G(0)/G(1) phase of cell cycle. Growth factors, such as insulin-like growth factor-I, epidermal growth factor and basic fibroblastic growth factor, markedly stimulated rBMSC proliferation in normal gravity, but had only a slight effect in simulated microgravity. Akt and extracellular signal-related kinase 1/2 phosphorylation levels and the expression of core-binding factor alpha1 decreased after 3 days of clinorotation culture. Microarray and gene ontology analyses further confirmed that rBMSC proliferation and osteogenesis decreased under simulated microgravity. CONCLUSIONS: The above data suggest that simulated microgravity inhibits population growth of rBMSCs and their differentiation towards osteoblasts. These changes may be responsible for some of the physiological changes noted during spaceflight.     

Common Effects on Cancer Cells Exerted by a Random Positioning Machine and a 2D Clinostat

In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML-1; RO82-W-1), which were exposed to a 2D clinostat (CLINO), a random positioning machine (RPM) and to normal 1g-conditions. After a three (3d)- or seven-day-culture (7d) on the two devices, we found both cell types growing three-dimensionally within multicellular spheroids (MCS) and also cells remaining adherent (AD) to the culture flask, while 1g-control cultures only formed adherent monolayers, unless the bottom of the culture dish was covered by agarose. In this case, the cytokines IL-6 and IL-8 facilitated the formation of MCS in both cell lines using the liquid-overlay technique at 1g. ML-1 cells grown on the RPM or the CLINO released amounts of IL-6 and MCP-1 into the supernatant, which were significantly elevated as compared to 1g-controls. Release of IL-4, IL-7, IL-8, IL-17, eotaxin-1 and VEGF increased time-dependently, but was not significantly influenced by the gravity conditions. After 3d on the RPM or the CLINO, an accumulation of F-actin around the cellular membrane was detectable in AD cells of both cell lines. IL-6 and IL-8 stimulation of ML-1 cells for 3d and 7d influenced the protein contents of ß₁-integrin, talin-1, Ki-67, and beta-actin dose-dependently in adherent cells. The ß₁-integrin content was significantly decreased in AD and MCS samples compared with 1g, while talin-1 was higher expressed in MCS than AD populations. The proliferation marker Ki-67 was elevated in AD samples compared with 1g and MCS samples. The ß-actin content of R082-W-1 cells remained unchanged. ML-1 cells exhibited no change in ß-actin in RPM cultures, but a reduction in CLINO samples. Thus, we concluded that simulated microgravity influences the release of cytokines in follicular thyroid cancer cells, and the production of ß₁-integrin and talin-1 and predicts an identical effect under real microgravity conditions.     

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252–267, 1998. © 1998 Wiley-Liss, Inc.     

Impact of microgravity on radiobiological processes and efficiency of DNA repair

To study the influence of microgravity on radiobiological processes in space, space experiments have been performed, using an on-board 1×g reference centrifuge as in-flight control. The trajectory of individual heavy ions was localized in relation to the biological systems by use of the Biostack concept, or an additional high dose of radiation was applied either before the mission or during the mission from an on-board radiation source. In embryonic systems, such as early developmental stages of Drosophila melanogaster and Carausius morosus, the occurrence of chromosomal translocations and larval malformations was dramatically increased in response to microgravity and radiation. It has been hypothesized that these synergistic effects might be caused by an interference of microgravity with DNA repair processes. However, recent studies on bacteria, yeast cells and human fibroblasts suggest that a disturbance of cellular repair processes in the microgravity environment might not be a complete explanation for the reported synergism of radiation and microgravity. As an alternative explanation, an impact of microgravity on signal transduction, on the metabolic/physiological state or on the chromatin structure at the cellular level, or modification of self-assembly, intercellular communication, cell migration, pattern formation or differentiation at the tissue and organ level should be considered.