Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) Hemocytes

Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground-based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.     

Cytosolic Calcium Concentration Changes in Neuronal Cells Under Clinorotation and in Parabolic Flight Missions

All life on earth has been established under conditions of stable gravity of 1g. Nevertheless, in numerous experiments the direct gravity dependence of biological processes has been shown on all levels of organization, from single molecules to humans. To study the effects especially of microgravity on biological systems, a variety of platforms are available, from drop towers to the ISS. Due to the costs of these platforms and their limited availability, as an alternative, numerous simulators have been developed for so called “simulated” microgravity. A classical systems is a clinostat, basically rotating a sample around one axis, and by integration of the gravity vector for 360° arguing that thus the effects of gravity are depleted. Indeed, a variety of studies has shown that taking out the direction of gravity from a biological system often results in consequences similar to the exposure of the system to real microgravity. Nevertheless, the opposite has been shown, too, and as a consequence the relevance of clinostats in microgravity research is still under discussion. To get some more insight into this problem we have constructed a small fluorescence clinostat and have studied the effects of clinorotation on the cytosolic calcium concentration of neuroglioma cells. The results have been compared to experiments with identical cells in real microgravity, utilizing parabolic flight missions. Our results show that in case of a cell suspension used in a small florescence clinostat within a tube diameter of 2mm, the effects of clinorotation are comparable to those under real microgravity, both showing a significant increase in intracellular calcium concentration.     

Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3^′,5^′-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤?0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.     

Comparative studies on gravisensitive protists on ground (2D and 3D clinostats) and in microgravity

In order to prepare and support space experiments, 2D and 3D clinostats are widely applied to study the influence of simulated weightlessness on biological systems. In order to evaluate the results a comparison between the data obtained in simulation experiments and in real microgravity is necessary. We are currently analyzing the gravity-dependent behavior of the protists Paramecium biaurelia (ciliate) and Euglena gracilis (photosynthetic flagellate) on these different experimental platforms. So far, first results are presented concerning the behaviour of Euglena on a 2D fast rotating clinostat and a 3D clinostat as well as under real microgravity conditions (TEXUS sounding rocket flight), of Paramecium on a 2D clinostat and in microgravity. Our data show similar results during 2D and 3D clinorotation compared to real microgravity with respect to loss of orientation (gravitaxis) of Paramecium and Euglena and a decrease of linearity of the cell tracks of Euglena. However, the increase of the mean swimming velocities, especially during 3D clinorotation (Euglena) and 2D clinorotation of Paramecium might indicate a persisting mechanostimulation of the cells. Further studies including long-term 2D and 3D clinostat exposition will enable us to demonstrate the qualification of the applied simulation methods.     

Microgravity performance of micro pulsating heat pipes

A micro pulsating heat pipe made of a thin clear Teflon tube of 1.6 mm ID was used to observe the pulsating flow inside a heat pipe under different gravity levels using parabolic flights. More vigorous pulsating flow was observed under microgravity, compared to the depressed movements under hypergravity. Two metallic micro pulsating heat pipes made of an aluminum plate with small internal channels were also tested to investigate the effect of gravity on their heat transfer characteristics. Reduced gravity experiments were performed aboard Falcon 20 aircraft flying parabolic trajectories. Under normal and hypergravity conditions, both the orientation of the pulsating heat pipe and locations of the heated and cooled sections affected the heat transfer performance. Under reduced gravity, however, the heat pipes showed better operating and heat transfer performance than that under normal and hypergravity. These experiments have for the first time confirmed that pulsating heat pipes are capable of operating under reduced gravity and thus are suitable for deployment in space applications such as satellites.     

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.     

Benefits of ESA Gravity-Related Hands-on Programmes for University Students’ Careers

The Education Office of the European Space Agency (ESA) offers university students, from ESA Member and Cooperating States, the opportunity to perform investigations in physical sciences, life sciences, and technology, under different gravity conditions through three educational programmes. The “Fly Your Thesis!” (FYT) programme makes use of parabolic flights and the “Drop Your Thesis!” (DYT) programme utilizes a drop tower as microgravity carriers, while the “Spin Your Thesis!” (SYT) programme uses a large centrifuge to create hypergravity. To date, more than hundred university students had the chance to participate in the design, development, and performance of one or more experiments during dedicated campaigns. In the following paper, we examine demographics of past participants of the ESA Education Office gravity-related opportunities over the past seven years and evaluate the benefits of these educational programmes for the participants’ studies and careers. Student teams that participated in one of the programmes between 2009 and 2013 were contacted to fill in a questionnaire. The feedback from the students demonstrate significant benefits extending far beyond the primary educational objectives of these programmes.     

Effects of fast clinostat treatment and microgravity on Vicia faba L. mesophyll cell protoplast ubiquitin pools and actin isoforms

With parabolic rocket flights and fast clinostat treatments, the effect of microgravity on ubiquitin, ubiquitin-protein conjugates, and actin isoforms of Vicia faba mesophyll protoplasts was studied. Western immunoblotting with ubiquitin antibodies revealed that simulated and particularly, real microgravity influenced the amount of free ubiquitin and of 18, 19, and 40 kD ubiquitin conjugates by inducing strong oscillations in the proteins concentrations over time. Simulated microgravity and microgravity-phase during parabolic rocket flights resulted in a decrease of actin isoforms. Results obtained support the assumption, that microgravity and fast clinostat treatment have a direct effect on Vicia faba mesophyll protoplast metabolic activities.     

Effects of Altered Gravity on the Cytoskeleton of Neonatal Rat Cardiocytes

As an intracellular load-bearing structure, the cytoskeleton is hypothesized to play a crucial role in gravity perception of the cell. Recent data show that the cytoskeleton, which includes actin microfilaments and microtubules, is involved in modulating both the electrical and the mechanical activities of the myocardium. The present study employed observation and quantified analyses of fluorescent images of cardiocytes under different gravity conditions. In acute gravitational change (micro- and hypergravity) induced by parabolic flight, we found disassembly of microtubules but enhanced polymerization of microfilaments, with rearrangement from G-actin to F-actin. In ground-based experiments, exposure of cardiocytes to 2×g hypergravity (centrifugation) led to increased width and number of actin fibers from 2 to 48 h, while microtubules showed no significant changes except polarization at 24 and 48 h. In contrast, exposure of cardiocytes to clinorotation led to disassembly of microtubules from 1 to 48 h, while microfilaments showed no significant changes except redistribution, which was accompanied by rounding of the cells (48 h). We assume that the sensitivity of microfilaments to hypergravity and that of microtubules to microgravity might contribute to the specific cytoskeletal changes observed in parabolic flight. These findings indicate different sensitivity and responses of microfilaments and microtubules to different gravitational changes, which might be part of functional adaptations of the cardiocytes to altered gravitational environments.     

Chemical oven technology and its application in AlN composites fabricated under microgravity condition

The microgravity experiments of fabrication materials by using TiC chemical ovens have been performed on the parabolic flight plane. The gravity behaviors in the combustion reactions of chemical ovens themselves during the aircraft parabolic flight were investigated. The results show that, the combustion temperatures and reactions vary with different gravity levels. These influences are related with the function of gravity on the molten titanium. As an example of the application of chemical ovens, the first and preliminary investigation of AlN-borosilicate glass composites fabricated in the chemical ovens during aircraft parabolic flights is conducted. The results indicate that, microgravity condition allows the synthesis of AlN-borosilicate glass composite with improved microstructure as compared with that on the ground.     

Effect of gravity on pool boiling heat transfer on thermal spray coating

This study deals with heat transfer enhancement surface manufactured by thermal spraying. Two thermal spraying methods using copper as a coating material, wire flame spraying (WFS) and vacuum plasma spraying (VPS), were applied to the outside of copper cylinder with 20 mm OD. The surface structure by WFS was denser than that by VPS. The effect of gravity on boiling heat transfer coeffcient and wall superheat at the onset of boiling were experimentally evaluated under micro- and hyper-gravity condition during a parabolic trajectory flight of an airplane. Pool boiling experiments in saturated liquid of HCFC123 were carried out for heat fluxes between 1.0 and 160 kW/m² and saturated temperature of 30 °C. As a result, the surface by VPS produced higher heat transfer coefficient and lower superheat at the onset of boiling under microgravity. For the smooth surface, the effect of gravity on boiling heat transfer coefficient was a little. For the coating, a large difference in heat transfer coefficient to gravity was observed in the moderate heat flux range. The heat transfer coefficinet decreased as gravity changed from the normal to hypergravity, and was improved as gravity changed from the hyperto microgravity. The difference in heat transfer coefficient between the normal and microgravity was a little. Heat transfer enhancement factor was kept over the experimental range of heat flux. It can be said that boiling behavior on thermal spray coating might be influenced by flow convection velocity.     

Gravitactic signal transduction elements in astasia longa investigated during parabolic flights

Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitactic behavior. Many experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism. The goal of the present study was to examine elements in the sensory transduction by means of inhibitors of gravitaxis and the intracellular calcium concentration during short microgravity periods. During the course of six parabolic flights (ESA 31th parabolic flight campaign and DLR 6th parabolic flight campaign) the effects of trifluoperazine (calmodulin inhibitor), caffeine (phosphodiesterase inhibitor) and gadolinium (blocks mechano-sensitive ion channels) was investigated. Due to the extreme parabolic flight maneuvers of the aircraft alternating phases of 1.8×g_n (about 20 s) and microgravity (about 22 s) were achieved (g_n: acceleration of Earth’s gravity field). The duration of the microgravity periods was sufficient to detect a loss of cell orientation in the samples. In the presence of gadolinium impaired gravitaxis was found during acceleration, while caffeine-treated cells showed, compared to the controls, a very precise gravitaxis and faster reorientation in the 1.8×g_n period following microgravity. A transient increase of the intracellular calcium upon increased acceleration was detected also in inhibitor-treated samples. Additionally, it was found that the cells showed a higher calcium signal when they deviated from the vertical swimming direction. In the presence of trifluoperazine a slightly higher general calcium signal was detected compared to untreated controls, while gadolinium was found to decrease the intracellular calcium concentration. In the presence of caffeine no clear changes of intracellular calcium were detected compared to the control. Dedicated to the memory of our colleague and friend Helmut Wagner     

Effects of Simulated Microgravity on Otolith Growth of Larval Zebrafish using a Rotating-Wall Vessel: Appropriate Rotation Speed and Fish Developmental Stage

Stimulus dependence is a general feature of developing animal sensory systems. In this respect, it has extensively been shown earlier that fish inner ear otoliths can act as test masses as their growth is strongly affected by altered gravity such as hypergravity obtained using centrifuges, by (real) microgravity achieved during spaceflight or by simulated microgravity using a ground-based facility. Since flight opportunities are scarce, ground-based simulators of microgravity, using a wide variety of physical principles, have been developed to overcome this shortcoming. Not all of them, however, are equally well suited to provide functional weightlessness from the perspective of the biosystem under evaluation. Therefore, the range of applicability of a particular simulator has to be extensively tested. Earlier, we have shown that a Rotating-Wall Vessel (RWV) can be used to provide simulated microgravity for developing Zebrafish regarding the effect of rotation on otolith development. In the present study, we wanted to find the most effective speed of rotation and identify the appropriate developmental stage of Zebrafish, where effects are the largest, in order to provide a methodological basis for future in-depth analyses dedicated to the physiological processes underlying otolith growth at altered gravity. Last not least, we compared data on the effect of simulated microgravity on the size versus the weight of otoliths, since the size usually is measured in related studies due to convenience, but the weight more accurately approximates the physical capacity of an otolith. Maintaining embryos at 10 hours post fertilization for three days in the RWV, we found that 15 revolutions per minute (rpm) yielded the strongest effects on otolith growth. Maintenance of Zebrafish staged at 10 hpf, 1 day post fertilization (dpf), 4 dpf, 7 dpf and 14 dpf for three days at 15 rpm resulted in the most prominent effects in 7 dpf larvae. Weighing versus measuring the size of otoliths yielded basically similar results, but the data gained by weighing were more distinct. Overall, our results clearly support the concept that the environmental gravity vector regulates fish otolith growth in terms of the pendulum model of otolith test masses, and that wall vessel rotation is a valuable means to provide functional weightlessness from the perspective of developing Zebrafish. We recommend that Zebrafish embryos staged 7 dpf (or possibly slightly elder) are rotated at 15 rpm in a Rotating-Wall Vessel as used in the present study for further experiments designed to elucidate the mechanisms underlying (altered gravity affected) otolith growth.     

Determination of the threshold of gravity for inducing kinetosis in fish: A drop-tower experiment

It has been repeatedly shown earlier that some fish of a given batch reveal motion sickness (a kinetosis) at the transition from 1 g to microgravity. In the course of parabolic aircraft flight experiments, it has been demonstrated that kinetosis susceptibility is correlated with asymmetric inner ear otoliths (i.e., differently weighed statoliths on the right and the left side of the head) or with genetically predispositioned malformed cells within the sensory epithelia of the inner ear. Hitherto, the threshold of gravity perception for inducing kinetotic behaviour as well as the relative importance of asymmetric otoliths versus malformed epithelia for kinetosis susceptibility has yet not been determined. The following experiment using the ZARM drop-tower facility in Bremen, Germany, is proposed to be carried out in order to answer the aforementioned questions. Larval cichlid fish (Oreochromis mossambicus) will be kept in a camcorder-equipped centrifuge during the microgravity phases of the drops and thus receive various gravity environments ranging from 0.1 to 0.9 g. Videographed controls will be housed outside of the centrifuge receiving 0 g. Based on the videorecordings, animals will be grouped into kinetotically and normally swimming samples. Subsequently, otoliths will be dissected and their size and asymmetry will be measured. Further investigations will focus on the numerical quantification of inner ear supporting and sensory cells as well as on the quantification of inner ear carbonic anhydrase reactivity. A correlation between (1) the results to be obtained concerning the g-loads inducing kinetosis and (2) the corresponding otolith asymmetry/morphology of sensory epithelia/carbonic anhydrase reactivity will further contribute to the understanding of the origin of kinetosis susceptibility. Besides an outline of the proposed principal experiments, the present study reports on a first series of drop-tower tests which were undertaken to elucidate the feasibility of the proposal (especially concerning the question, if some 4.7 s of microgravity are sufficient to induce kinetotic behaviour in larval fish).     

Real-Time Video-Microscopy of Migrating Immune Cells in Altered Gravity During Parabolic Flights

In our project we developed a technical equipment which allows to visualize migration of cells in real-time video-microscopy during altered gravity conditions of NOVESPACE Airbus A300 ZERO-G parabolic flights. For validation of the experimental device we have used fast moving human neutrophils as example, because their migration is fundamental to keep the organism under immunological surveillance. Their migration is indispensable for immune effector function, where the cells leave the blood vessels and navigate to places of infection to fulfill their main task of phagocytosis. Thereby, we have analyzed if their migration is affected during altered gravity conditions and if pharmacological modification of cytoskeletal dynamics influences neutrophil migratory activity. Whereas we detected no change in neutrophil locomotory behaviour in microgravity, we found a significant inhibitory influence of hypergravity, irrespective of the chemical stimulus used. Our results suggest that hypergravity, following a microgravity environment, could represent a hazard to the human immune system function. Thus, our cell migration assay offers an optimum experimental device for studying the migratory activity and underlying signal transduction mechanisms of neutrophils to assess the immunological fitness of humans in space to fight infection, but also for investigating the locomotion of other cell types or unicellular organisms such as ciliates.     

Vegetative growth of higher plants on a three-dimensional clinostat

Seedlings of rice, maize, cress, pea, and azuki bean were grown on a three-dimensional clinostat and changes in their vegetative growth processes were analyzed. A balanced relationship among the length or the weight of each organ was observed in these species even on the clinostat. Growth of pea second internodes is supported by the transport of sugars from the cotyledons, which was not influenced by the clinostat rotation. Thus, growth correlation and the translocation of sugars normally occurred even under simulated microgravity conditions. In contrast, morphogenesis was clearly changed by the clinostat rotation. The axiality along the gravity vector disappeared and so seedlings formed themselves into a sphere-like shape on the clinostat. The dorsiventrality was indistinct in growth of maize coleoptiles on the surface of the earth, but the clinostat rotation induced a clear dorsinventral bending. These changes in morphogenesis may influence the long-term growth phenomena and modify the life cycle of higher plants under a microgravity environment.     

Clinorotation Increases the Growth of Utricular Otoliths of Developing Cichlid Fish

It has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish as an adaptation towards increased environmental gravity. Suggesting that otolith growth is regulated by the central nervous system, thus adjusting otolithic weight to produce a test mass, applying functional weightlessness should yield an opposite effect, i.e. larger than normal otoliths. Therefore, larval siblings of cichlid fish (Oreochromis mossambicus) were housed for 7 days in a submersed, two-dimensional clinostat, which provided a residual gravity of approximately 0.007g. After the experiment, otoliths were dissected and their size (area grown during the experiment) was determined. Maintenance in the clinostat resulted in significantly larger utricular otoliths (lapilli, involved in graviperception). There were no statistical significant differences regarding saccular otoliths obtained (sagittae, involved in transmitting linear acceleration and, especially, in the hearing process). These results indicated, that the animals had in fact received functional weightlessness. In line and contrasting results on the otoliths of other teleost species kept at actual microgravity (spaceflight) or within rotating wall vessels are discussed.     

Tropistic responses of Avena seedlings in simulated hypogravity

Results from investigation of the gravitropic responses of the Avena coleoptile under simulated weightlessness are presented. The tests were conducted using the flight hardware, identified by NASA as the Gravitational Plant Physiology Facility (GPPF), designed to support the Spacelab experiment, GTHRES. Weightlessness or so-called microgravity-conditions were simulated by the use of a so-called somersault clinostat. The plants were gravitropically stimulated with different g-forces and stimulation durations on a centrifuge. Timelapse video pictures of the plants were taken during the last hour prior to the stimulation and in a period of 3 hours after. The gravitropic responses of the plants were analyzed with the aid of an image analyzing system. Dose-response curves for stimulation forces 0.2, 0.4, 0.6, 0.8, and 1.0 g with different stimulation times ranging from 2 to 250 min were achieved. The results show that the reciprocity between force and stimulation time may be valid for small stimulation doses (lower than 5 g min), but not for larger doses. Furthermore, the threshold values (i.e. the smallest stimulation the plants are able to detect) found by extrapolation of the dose response curves, are less than 30 g s. The results are discussed with respect to an accepted space experiment, GTHRES, where the corresponding experiments will be carried out in the same apparatus under true microgravity conditions.     

Gravity effect on spray impact and spray cooling

An experimental study of the film produced by the spray impact on a heated target and of the spray cooling has been performed in normal gravity and in microgravity conditions during parabolic flights. A convex shape of the target allowed visualization of the film evolution and determination of the film characteristics using the image processing. The effects of the spray parameters and of the gravity level on heat transfer have been investigated. It has been found that the spray cooling efficiency depends on the water flow rate in a non-monotonous way. A range of spray parameters at which the effect of gravity level on heat transfer is significant has been determined. It has been found that the spray cooling is less effective in microgravity conditions in comparison with normal gravity and hypergravity.     

Development of a 3He Refrigerator for Possible Experiments of Solid 4He on a Small Jet Plane

The gravity on the Earth (g _E) has not been taken seriously to mask the fundamental phenomena on quantum solids, though there are some important studies on the critical phenomena of superfluid ⁴He under microgravity. We are planning to investigate the effect of gravity on the equilibrium shape of solid ⁴He. Since we had a chance to do such an experiment on a small jet plane through the ground based program by JAXA, we got started to construct a cryostat which could cool down as low as 500?mK and meet severe restrictions of experiments on a jet plane. The main part of the refrigerator was a usual ³He-evaporator pumped by a scroll pump. A?small GM refrigerator was installed to provide 4?K stage. 1?K pot was also put in which was also pumped by another scroll pump to condense ³He gas and sample ⁴He. The cryostat was designed to have two optical windows to be able to observe solid ⁴He under microgravity. In the test flight for the refrigerator, the minimum temperature of 690?mK was kept during the entire flight of two hours in which 7 to 8 times parabolic flight was performed. Each parabolic flight includes about 20?seconds microgravity and 20?seconds 1.5 to 2.0?g _E period before and after the microgravity. We did some preliminary experiments with bcc solid ⁴He under microgravity. The crystal remained stuck to the bottom of the sample cell even in the 20 seconds microgravity condition.