Longevity in space; experiment on the life span of Paramecium cell clone in space.

Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described.     

Longevity in space; Experiment on the life span of Paramecium cell clone in space

Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described.     

Cell fusion in space: plasma membrane fusion in human fibroblasts during short term microgravity.

During short-term microgravity in sounding rocket experiments (6 min.) the cytoskeleton undergoes changes and therefore it is possible that cell processes which are dependent on the structure and function of the cytoskeleton are influenced. A cell fusion experiment, initiated by a short electric pulse, was chosen as a model experiment for this sounding rocket experiment. Confluent monolayers of primary human skin fibroblasts, grown on coverslips, were mounted between two electrodes (distance 0.5 cm) and fused by discharging a capacitor (68 micro F; 250 V; 10 msec) in a low conductive medium. During a microgravity experiment in which nearly all the requirements for an optimal result were met (only the recovery of the payload was delayed) results were found that indicated that microgravity during 6 minutes did not influence cell fusion since the percentage of fused products did not change during microgravity. Within the limits of discrimination using morphological assays microgravity has no influence on the actin/cortical cytoskeleton just after electrofusion.     

微重力环境中哺乳动物细胞分子生物学效应研究进展

随着载人航天事业的不断发展,空间失重环境引起的航天员健康问题（心血管疾病、免疫抑制、肌肉萎缩、骨质疏松等）日益突出,这已成为人类探索空间的一大阻碍.越来越多的研究关注到微重力条件下机体及细胞的变化.近期的研究表明,在细胞水平上,微重力会引起细胞降解,改变细胞骨架,并造成细胞在分子水平（如细胞增殖、分化、迁移、粘附、信号转导等过程）的一系列改变.本文对微重力条件下免疫细胞、内皮细胞、骨细胞、癌细胞的相关研究进行了归纳总结,研究结果可为微重力条件下机体及相关细胞的研究提供指导,为治疗或缓解微重力条件造成的疾病提供方法和思路.      

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.     

Development of an improved ground-based prototype of space plant-growing facility

Based on a formerly developed ground-based prototype of space plant-growing facility, the development of its improved prototype has been finished, so as to make its operating principle better adapt to the space microgravity environment. According to the developing experience of its first generation prototype and detailed demonstration and design of technique plan, its blueprint design and machining of related components, whole facility installment, debugging and trial operations were all done gradually. Its growing chamber contains a volume of about 0.5 m³ and a growing area of approximate 0.5 m²; the atmospheric environmental parameters in the growing chamber and water content in the growing media were controlled totally and effectively; lighting source is a combination of both red and blue light emitting diodes (LED). The following demonstrating results showed that the entire system design of the prototype is reasonable and its operating principle can nearly meet the requirements of space microgravity environment. Therefore, our plant-growing technique in space was advanced further, which laid an important foundation for next development of the space plant-growing facility and plant-cultivating experimental research in space microgravity condition.     

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.     

Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration.

Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.     

Simulated microgravity does not alter epithelial cell adhesion to matrix and other molecules.

Microgravity has advantages for the cultivation of tissues with high fidelity; however, tissue formation requires cellular recognition and adhesion. We tested the hypothesis that simulated microgravity does not affect cell adhesion. Human colorectal carcinoma cells were cultured in the NASA Rotating Wall Vessel (RWV) under low shear stress with randomization of the gravity vector that simulates microgravity. After 6-7 days, cells were assayed for binding to various substrates and compared to cells grown in standard tissue culture flasks and static suspension cultures. The RWV cultures bound as well to basement membrane proteins and to CEA, an intercellular adhesion molecule, as control cultures did. Thus, microgravity does not alter epithelial cell adhesion and may be useful for tissue engineering.     

Simulated microgravity inhibits the proliferation of K562 erythroleukemia cells but does not result in apoptosis

Astronauts and experimental animals in space develop the anemia of space flight, but the underlying mechanisms are still unclear. In this study, the impact of simulated microgravity on proliferation, cell death, cell cycle progress and cytoskeleton of erythroid progenitor-like K562 leukemia cells was observed. K562 cells were cultured in NASA Rotary Cell Culture System (RCCS) that was used to simulate microgravity (at 15 rpm). After culture for 24 h, 48 h, 72 h, and 96 h, the cell densities cultured in RCCS were only 55.5%, 54.3%, 67.2% and 66.4% of the flask-cultured control cells, respectively. The percentages of trypan blue-stained dead cells and the percentages of apoptotic cells demonstrated no difference between RCCS-cultured cells and flask-cultured cells at every time points (from 12 h to 96 h). Compared with flask-cultured cells, RCCS culture induced an accumulation of cell number at S phase concomitant with a decrease at G0/G1 and G2/M phases at 12 h. But 12 h later (from 24 h to 60 h), the distribution of cell cycle phases in RCCS-cultured cells became no difference compared to flask-cultured cells. Consistent with the changes of cell cycle distribution, the levels of intercellular cyclins in RCCS-cultured cells changed at 12 h, including a decrease in cyclin A, and the increasing in cyclin B, D1 and E, and then (from 24 h to 36 h) began to restore to control levels. After RCCS culture for 12–36 h, the microfilaments showed uneven and clustered distribution, and the microtubules were highly disorganized. These results indicated that RCCS-simulated microgravity could induce a transient inhibition of proliferation, but not result in apoptosis, which could involve in the development of space flight anemia. K562 cells could be a useful model to research the effects of microgravity on differentiation and proliferation of hematopoietic cells.     

空间高等植物培养装置

空间高等植物培养装置用于中国天宫二号空间实验室开展微重力条件下高等植物生长机理研究.该装置由高等植物培养模块、生命保障模块、实时在线检测模块和返回单元等功能单元组成,可实现高等植物空间长周期培养,在轨启动生物实验,实时在线观察和荧光监测,水分循环利用及营养供给,模拟太阳长短日照周期控制与检测,环境温度测量与控制,CO₂浓度调节,有害气体去除及航天员回收部分样品等功能.      

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.     

Possible mechanisms of plant cell wall changes at microgravity.

Space and clinostatic experiments revealed that changes of plant cell wall structure and its function depend on type of tissue and duration of influence. It was shown that clinostat conditions reproduce the part of weightlessness biological effects. It is established that various responses of wall structural-metabolic organization occur at microgravity: changes of cell walls ultrastructure and organelles structure; decrease of synthesis of primary plant cell wall; rearrangements of polysaccharides content. It is shown that mechanisms of plant cell wall changes at microgravity are connected with decrease of cellulose crystallization, activation of pectolytic enzymes and rearrangement of calcium balance of apoplast and cytoplasm.     

Space flight, microgravity, stress, and immune responses.

Exposure of animals and humans to space flight conditions has resulted in numerous alterations in immunological parameters. Decreases in lymphocyte blastogenesis, cytokine production, and natural killer cell activity have all been reported after space flight. Alterations in leukocyte subset distribution have also been reported after flight of humans and animals in space. The relative contribution of microgravity conditions and stress to the observed results has not been established. Antiorthostatic, hypokinetic, hypodynamic, suspension of rodents and chronic head-down tilt bed-rest of humans have been used to model effects of microgravity on immune responses. After use of these models, some effects of space flight on immune responses, such as decreases in cytokine function, were observed, but others, such as alterations in leukocyte subset distribution, were not observed. These results suggest that stresses that occur during space flight could combine with microgravity conditions in inducing the changes seen in immune responses after space flight. The biological/biomedical significance of space flight induced changes in immune parameters remains to be established. Grant Numbers: NCC2-859, NAG2-933.     

Farming in space: environmental and biophysical concerns.

The colonization of space will depend on our ability to routinely provide for the metabolic needs (oxygen, water, and food) of a crew with minimal re-supply from Earth. On Earth, these functions are facilitated by the cultivation of plant crops, thus it is important to develop plant-based food production systems to sustain the presence of mankind in space. Farming practices on earth have evolved for thousands of years to meet both the demands of an ever-increasing population and the availability of scarce resources, and now these practices must adapt to accommodate the effects of global warming. Similar challenges are expected when earth-based agricultural practices are adapted for space-based agriculture. A key variable in space is gravity; planets (e.g. Mars, 1/3 g) and moons (e.g. Earth's moon, 1/6 g) differ from spacecraft orbiting the Earth (e.g. Space stations) or orbital transfer vehicles that are subject to microgravity. The movement of heat, water vapor, CO2 and O2 between plant surfaces and their environment is also affected by gravity. In microgravity, these processes may also be affected by reduced mass transport and thicker boundary layers around plant organs caused by the absence of buoyancy dependent convective transport. Future space farmers will have to adapt their practices to accommodate microgravity, high and low extremes in ambient temperatures, reduced atmospheric pressures, atmospheres containing high volatile organic carbon contents, and elevated to super-elevated CO2 concentrations. Farming in space must also be carried out within power-, volume-, and mass-limited life support systems and must share resources with manned crews. Improved lighting and sensor technologies will have to be developed and tested for use in space. These developments should also help make crop production in terrestrial controlled environments (plant growth chambers and greenhouses) more efficient and, therefore, make these alternative agricultural systems more economically feasible food production systems.     

Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.

We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.     

Thin film bioreactors in space.

Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization and cellular differentiation. Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers us an opportunity to learn more about basic biological systems with one important variable removed. The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions. Studies on cell cultures grown in microgravity would enable us to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.     

In vitro plant cell growth in microgravity and on clinostat.

For the study of gravity's role in the processes of plant cell differentiation in-vitro, a model "seed-seedling-callus" has been used. Experiments were carried out on board the orbital stations Salyut-7 and Mir as well as on clinostat. They lasted from 18 to 72 days. It was determined that the exclusion of a one-sided action of gravity vector by means of clinostat and spaceflight conditions does not impede the formation and growth of callus tissue; however, at cell and subcellular levels structural and functional changes do take place. No significant changes were observed either on clinostat or in space concerning the accumulation of fresh biomass, while the percentage of dry material in space is lower than in control. Both in microgravity (MG) and in control, even after 72 days of growth, cells with a normally developed ultrastructure are present. In space, however, callus tissue more often contains cells in which the cross-section area of a cell, a nuclei and of mitochondria are smaller and the vacuole area--bigger than in controls. In microgravity a considerable decrease in the number of starch-containing cells and a reduction in the mean area of starch grains in amyloplasts is observed. In space the amount of soluble proteins in callus tissue is 1.5 times greater than in control. However, no differences were observed in fractions when separated by the SDS-PAGE method. In microgravity the changes in cell wall material components was noted. In the space-formed callus changes in the concentration of ions K, Na, Mg, Ca and P were observed. However, the direction of these changes depends on the age of callus. Discussed are the possible reasons for modification of morphological and metabolic parameters of callus cells when grown under changed gravity conditions.     

微重力对拟南芥长势影响的代谢网络流平衡分析

微重力作为典型的空间环境因素,对植物生长发育的影响机制是空间生命科学的研究热点。微重力环境直接或间接影响植物代谢,并引起许多生理适应。 随着系统生物学的发展,代谢网络模型使微重力环境下的植物代谢建模成为可能。采用流平衡分析方法对模式植物拟南芥不同组织的代谢网络进行分析,研究微重力对拟南芥生长发育的影响机制。通过比较空间与地面条件下拟南芥的生物质产量,发现空间条件下拟南芥黄化幼苗、幼苗、芽、根、下胚轴的生物量分别下降了33.00%,51.52%,6.89%,12.53%,11.70%,与空间环境下拟南芥的长势变化趋势一致。代谢通路富集分析发现,微重力使得拟南芥的碳固定等通路下调,而磷酸戊糖途径上调,初步解析了微重力对拟南芥生长发育的影响机制,也验证了流平衡方法用于微重力生物学效应研究中的可行性。      

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.