The “<Emphasis Type="Italic">Daphnia</Emphasis>” Lynx Mark I Suborbital Flight Experiment: Hardware Qualification at the Drop Tower Bremen

The Drop Tower Bremen, a ground-based facility enabling research under real microgravity conditions, is an excellent platform for testing new types of experimental hardware to ensure full performance when deployed in costly and rare flight opportunities such as suborbital flights. Here we describe the “Daphnia” experiment which will fly on XCOR Aerospace Lynx Mark I and our experience from the hardware tests with the catapult system at the drop tower. The aim of the “Daphnia” experiment is to obtain data on the biological performance of daphnids and predator-prey interactions in microgravity, which are important for the development of aquatic bioregenerative life support systems (BLSS). The experiment consists of two subunits: The first unit is dedicated to predator-prey interactions, where behavioural analysis should reveal if microgravity interfere with prey (Daphnia) detection or feeding and therefore may interrupt the trophic cascade. The functioning of such an artificial food web is indispensable for a long-lasting BLSS suitable for long-duration manned space missions or Earth-based explorations to extreme habitats. The second unit is designed to investigate the impact of microgravity on gene expression and the cytoskeleton in Daphnia. Next to data collection, the real microgravity conditions at the drop tower have helped to identify the weak points of the “Daphnia” experimental hardware and lead to further improvement. Hence, the drop tower is ideal for testing new experimental hardware which is indispensable before the implementation in suborbital flights.     

Tissue Engineering of Cartilage on Ground-Based Facilities

Investigations under simulated microgravity offer the opportunity for a better understanding of the influence of altered gravity on cells and the scaffold-free three-dimensional (3D) tissue formation. To investigate the short-term influence, human chondrocytes were cultivated for 2 h, 4 h, 16 h, and 24 h on a 2D Fast-Rotating Clinostat (FRC) in DMEM/F-12 medium supplemented with 10 % FCS. We detected holes in the vimentin network, perinuclear accumulations of vimentin after 2 h, and changes in the chondrocytes shape visualised by F-actin staining after 4 h of FRC-exposure. Scaffold-free cultivation of chondrocytes for 7 d on the Random Positioning Machine (RPM), the FRC and the Rotating Wall Vessel (RWV) resulted in spheroid formation, a phenomenon already known from spaceflight experiments with chondrocytes (MIR Space Station) and thyroid cancer cells (SimBox/Shenzhou-8 space mission). The experiments enabled by the ESA-CORA-GBF programme gave us an optimal opportunity to study gravity-related cellular processes, validate ground-based facilities for our chosen cell system, and prepare long-term experiments under real microgravity conditions in space     

Germination of Arabidopsis Seed in Space and in Simulated Microgravity: Alterations in Root Cell Growth and Proliferation

Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions.     

Responses of Microcrustaceans to Simulated Microgravity (2D-Clinorotation) - Preliminary Assessments for the Development of Bioregenerative Life Support Systems (BLSS)

Bioregenerative Life Support Systems (BLSS) are an endeavor to create environments able to maintain human life e.g. on future long-duration space missions like flights to Mars. Based on cyclic biological processes, these systems will be independent from material resupply (such as food, water and oxygen). Due to their central role in limnic ecosystems, herbivorous microcrustaceans could act as key player in aquatic BLSS as they link oxygen liberating, autotrophic producers like algae to higher trophic levels, such as fish. However, before such BLSS can be utilized in space, organisms inhabiting these systems have to be studied thoroughly to disclose the gravitational impact on the biological processes. This is possible in real microgravity, but requires high financial resources, is opportunity-limited or periods of microgravity are very short. Yet, cost-effective and almost permanently accessible tools for gravitational research are ground-based facilities (GBFs), providing simulated microgravity. Among those GBFs is the so called 2D-clinostat. In the present study we demonstrate, that rotation of clinostat tubes does not generate acceleration in form of (predator resembling) small scale turbulence, which can be perceived by Daphnia cucullata. Additionally, embryonal development is not disturbed in subitaneous eggs of Daphnia magna and resting eggs of the ostracod Heterocypris incongruens (besides through restrictions in space within the narrow clinostat tubes), just as subsequent hatching from the respective eggs. Hence, our results indicate that clinorotation is a suitable method to simulate microgravity for microcrustaceans.     

Thyroid Cells Exposed to Simulated Microgravity Conditions – Comparison of the Fast Rotating Clinostat and the Random Positioning Machine

The ground-based facilities 2D clinostat (CN) and Random Positioning Machine (RPM) were designed to simulate microgravity conditions on Earth. With support of the CORA-ESA-GBF program we could use both facilities to investigate the impact of simulated microgravity on normal and malignant thyroid cells. In this review we report about the current knowledge of thyroid cancer cells and normal thyrocytes grown under altered gravity conditions with a special focus on growth behaviour, changes in the gene expression pattern and protein content, as well as on altered secretion behaviour of the cells. We reviewed data obtained from normal thyrocytes and cell lines (two poorly differentiated follicular thyroid cancer cell lines FTC-133 and ML-1, as well as the normal thyroid cell lines Nthy-ori 3-1 and HTU-5). Thyroid cells cultured under conditions of simulated microgravity (RPM and CN) and in Space showed similar changes with respect to spheroid formation. In static 1g control cultures no spheroids were detectable. Changes in the regulation of cytokines are discussed to be involved in MCS (multicellular spheroids) formation. The ESA-GBF program helps the scientists to prepare future spaceflight experiments and furthermore, it might help to identify targets for drug therapy against thyroid cancer.     

Rocket seedling production on the international space station: Growth and nutritional properties

Producing sprouts directly during space missions may represent an interesting opportunity to offer high-quality fresh ready to eat food to the astronauts. The goal of this work was to compare, in terms of growth and nutritional quality, rocket (Eruca sativa Mill.) seedlings grown in the International Space Station during the ENEIDE mission with those grown in a ground-based experiment (in presence and absence of clinorotation). The rocket seedlings obtained from the space-experiment were thinner and more elongated than those obtained in the ground-based experiment. Cotyledons were often closed in the seedlings grown in the space experiment. Quantitative (germination, fresh and dry weight) and qualitative (glucose, fructose, sucrose and starch) traits of rocket seedling were negatively affected by micrograv-ity, especially those recorded on seedlings grown under real microgravity conditions The total chlorophyll, and carotenoids of seedlings obtained in the space experiment were strongly reduced in comparison to those obtained in the ground-based experiment (presence and absence of clinorotation). The results showed that it is possible to produce rocket seedlings in the ISS; however, further studies are needed to define the optimal environmental conditions for producing rocket seedlings with high nutritional value     

Gasless combustion-driven heating elements for materials experiments in space

The novel concept of using gasless combustible mixtures as heating elements for materials processing in space and in ground-based microgravity facilities is presented. The unique properties of metal-sulfur combustible compositions (i.e., high flame temperatures, low ignition temperatures, liquid combustion products, nonporous charges, and gasless reactions) make them ideally suited for such heating applications. Heating elements based on metal-sulfur combustion have an energy density more than order of magnitude greater than electrical batteries, can be easily integrated with processing samples, and can operate under high pressures and in different gaseous environments. Demonstration prototypes of the gasless combustion-driven furnaces have already demonstrated peak temperatures close to 2300 K and heating rates above 200 °C/s.     

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.     

The Impact of Hypergravity and Vibration on Gene and Protein Expression of Thyroid Cells

Experiments in space either on orbital missions on-board the ISS, or in suborbital missions using sounding rockets, like TEXUS as well as parabolic flight campaigns are still the gold standard to achieve real microgravity conditions in the field of gravitational biology and medicine. However, during launch, and in flight, hypergravity and vibrations occur which might interfere with the effects of microgravity. It is therefore important to know these effects and discriminate them from the microgravity effects. This can be achieved by ground-based facilities like centrifuges or vibration platforms. Recently, we have conducted several experiments with different thyroid cancer cell lines. This study, as part of the ESA-CORA-GBF 2010-203 project, focused on the influence of vibration and hypergravity on benign human thyroid follicular epithelial cells (Nthy-ori 3-1 cell line). Gene and in part protein expression regulation under both conditions were analyzed for VCAN, ITGA10, ITGB1, OPN, ADAM19, ANXA1, TNFA, ABL2, ACTB, PFN2, TLN1, EZR, RDX, MSN, CTGF, PRKCA, and PRKAA1 using quantitative real-time PCR and Western Blot. We found that hypergravity and vibration affected genes and proteins involved in the extracellular matrix, the cytoskeleton, apoptosis, cell growth and signaling. Vibration always led to a down-regulation, whereas hypergravity resulted in a more heterogeneous expression pattern. Overall we conclude that both conditions can influence gene regulation and production of various genes and proteins. As a consequence, it is important to perform control experiments on hypergravity and vibration facilities in parallel to flight experiments.     

基于悬吊法和气浮法的多自由度微重力模拟展开试验系统研究

为满足三维空间展开机械臂在进行地面微重力模拟展开试验时对多维度、多自由度、高精度展开轨迹和高卸载效率的要求,通过对当前成熟的微重力模拟展开试验方法进行分析总结,设计了一种基于悬吊法和气浮法的多自由度微重力模拟展开试验系统。首先,对所设计的多自由度微重力模拟展开试验系统进行结构设计和原理分析;然后,将多自由度微重力模拟展开试验系统应用于某三维空间展开机械臂的三维轨迹微重力模拟展开试验。结果表明该三维空间展开机械臂展开过程稳定可靠,且微重力模拟展开试验系统对机械臂产生的气浮运动摩擦阻力、垂直方向阻力波动量和展开方向附加阻力均很小,卸载效率高于95%,满足高精度和高卸载效率的展开要求。研究结果可为多自由度空间可展开机构地面模拟展开试验系统的深入研究提供一定的参考。     

Review of bubble detector response characteristics and results from space

A passive neutron-bubble dosemeter (BD), developed by Bubble Technology Industries, has been used for space applications. Both the bubble detector-personal neutron dosemeter and bubble detector spectrometer have been studied at ground-based facilities in order to characterise their response due to neutrons, heavy ion particles and protons. This technology was first used during the Canadian-Russian collaboration aboard the Russian satellite BION-9, and subsequently on other space missions, including later BION satellites, the space transportation system, Russian MIR space station and International Space Station. This paper provides an overview of the experiments that have been performed for both ground-based and space studies in an effort to characterise the response of these detectors to various particle types in low earth orbit and presents results from the various space investigations.     

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.     

IDR/UPM facilities for liquid bridge experimentation on Earth under microgravity conditions

Besides space laboratories for in-orbit experimentation, Earth based facilities for laboratory experimentation are of paramount importance for the enhancement on liquid bridge knowledge. In spite of the constraints imposed by simulated microgravity (which force to work either with very small size liquid bridges or by using the Plateau tank technique, amongst other techniques), the availability and accessibility of Earth facilities can circumvent in many cases the drawbacks associated with simulated microgravity conditions. To support theoretical and in orbit experimental studies on liquid bridges under reduced gravity conditions, several ground facilities were developed at IDR. In the following these ground facilities are briefly described, and main results obtained by using them are cited.     

A Novel Microgravity Simulator Applicable for Three-Dimensional Cell Culturing

Random Positioning Machines (RPM) were introduced decades ago to simulate microgravity. Since then numerous experiments have been carried out to study its influence on biological samples. The machine is valued by the scientific community involved in space relevant topics as an excellent experimental tool to conduct pre-studies, for example, before sending samples into space. We have developed a novel version of the traditional RPM to broaden its operative range. This novel version has now become interesting to researchers who are working in the field of tissue engineering, particularly those interested in alternative methods for three-dimensional (3D) cell culturing. The main modifications concern the cell culture condition and the algorithm that controls the movement of the frames for the nullification of gravity. An incubator was integrated into the inner frame of the RPM allowing precise control over the cell culture environment. Furthermore, several feed-throughs now allow a permanent supply of gas like CO ₂. All these modifications substantially improve conditions to culture cells; furthermore, the rewritten software responsible for controlling the movement of the frames enhances the quality of the generated microgravity. Cell culture experiments were carried out with human lymphocytes on the novel RPM model to compare the obtained response to the results gathered on an older well-established RPM as well as to data from space flights. The overall outcome of the tests validates this novel RPM for cell cultivation under simulated microgravity conditions.     

JAXA-GCF project - high-quality protein crystals grown under microgravity environment for better understanding of protein structure

Since 2003, Japan Aerospace Exploration Agency (JAXA, former NASDA) has been conducting a project on a semi-annual basis (JAXA-GCF) to obtain high-quality protein crystals in the microgravity environment using the Russian transportation system. For this project, protein samples were mostly provided by Japanese users for whom JAXA provided technical and clerical support for crystallization experiments in microgravity. For the project, JAXA has constructed a user-friendly support service for microgravity experiments and provided regular and frequent flight opportunities. To simplify and improve technological matters, JAXA devised a gel-tube method crystallization device, which is effective both in space and on ground, based on the counter-diffusion technique. JAXA also provided ground-based techniques for efficient preliminary optimization of crystallization conditions using a 1-dimensional simulation and for harvesting and cryoprotecting crystals before X-ray diffraction experiments. These improvements have significantly increased the success rate of obtaining useful results. In conclusion, JAXA has developed technologies for growing, in microgravity, high-quality protein crystals, which may diffract up to atomic resolution, for a better understanding of 3-dimensional protein structures through X-ray diffraction experiments.     

CECILIA,a versatile research tool for cellular responses to gravity

We describe a centrifuge designed and constructed according to current demands for a versatile instrument in cellular gravitational research, in particular protists (ciliates, flagellates). The instrument (called CECILIA,centrifuge forciliates) is suited for videomonitoring, videorecording, and quantitative evaluation of data from large numbers of swimming cells in a ground-based laboratory or in a drop tower/drop shaft under microgravity conditions. The horizontal rotating platform holds up to six 8mm-camcorders and six chambers holding the experimental cells. Under hypergravity conditions (up to 15 g) chambers can be rotated about 2 axes to adjust the swimming space at right angles or parallel to the resulting gravity vector. Evaluations of cellular responses to central acceleration — in the presence of gravitational 1 g — are used for extrapolation of cellular behaviour under hypogravity conditions. CECILIA is operated and monitored by computer using a custom-made soft-ware. Times and slopes of rising and decreasing acceleration, values and quality of steady acceleration are supervised online. CECILIA can serve as an on-ground research instrument for precursor investigations of the behaviour of ciliates and flagellates under microgravity conditions such as long-term experiments in the International Space Station.     

Embedding Arabidopsis Plant Cell Suspensions in Low-Melting Agarose Facilitates Altered Gravity Studies

Gravity plays a role in modulating plant growth and development and its alteration induces changes in these processes. Microgravity research has recently been extended to the use of in vitro plant cell cultures which are considered as an ideal model system to study cell proliferation and growth. In general, among the ground-based facilities available for microgravity simulation, the 2D pipette clinostat had been previously considered a suitable facility to be used for unicellular biological models although studies using single plant cell cultures raised some concerns. The incompatibility comes from the standard requirement of shaking a suspension culture for assuring its viability and active proliferation status in the control samples. Moreover, a related issue applies to the use of the random positioning machine (RPM) for cell suspension experiments. Here, we demonstrate an alternative culture method based on the immobilization of the culture before the altered gravity treatment occurs, such that it behaves as a solid object. Our immobilization procedure preserved plant cell culture viability without compromising basic cell properties as viability, morphology, cell cycle phases distribution, or chromatin organization, when compared with a standard cell suspension under shaking as a control. This approach should allow the space biology community to improve the quantity and quality of plant cell results in future simulated microgravity experiments or spaceflight opportunities.     

Transport of microparticles in weakly ionized gas-discharge plasmas under microgravity

Effective structural (pair correlation function) and transport (diffusion constant) characteristics of the system of microparticles (dusty plasma) have been measured in a set of experiments in dc- and rf gasdischarge plasmas in ground-based and microgravity conditions on the board of Russian “Mir” Space Station and International Space Station. The comparison between these measurements and numerical simulations were used for dusty plasma diagnostics. Specifically, the effective coupling parameter, which mainly governs structural and dynamical properties of the system, was determined for different experimental conditions. The application of these measurements to estimate the particle charge and the plasma screening length is discussed.     

CECILIA, a versatile research tool for cellular responses to gravity

We describe a centrifuge designed and constructed according to current demands for a versatile instrument in cellular gravitational research, in particular protists (ciliates, flagellates). The instrument (called CECILIA,centrifuge forciliates) is suited for videomonitoring, videorecording, and quantitative evaluation of data from large numbers of swimming cells in a ground-based laboratory or in a drop tower/drop shaft under microgravity conditions. The horizontal rotating platform holds up to six 8mm-camcorders and six chambers holding the experimental cells. Under hypergravity conditions (up to 15 g) chambers can be rotated about 2 axes to adjust the swimming space at right angles or parallel to the resulting gravity vector. Evaluations of cellular responses to central acceleration — in the presence of gravitational 1 g — are used for extrapolation of cellular behaviour under hypogravity conditions. CECILIA is operated and monitored by computer using a custom-made soft-ware. Times and slopes of rising and decreasing acceleration, values and quality of steady acceleration are supervised online. CECILIA can serve as an on-ground research instrument for precursor investigations of the behaviour of ciliates and flagellates under microgravity conditions such as long-term experiments in the International Space Station.     

From waste to energy: First experimental bacterial fuel cells onboard the international space station

Bacterial Fuel Cells are innovative energy systems that use bacteria to transform carbohydrates anaerobically into free electrons and waste products. The bacteria deposit the electrons on the anode and hence create a potential difference between the anode and the cathode, yielding a ‘bacterial battery’. This principle may be favourably influenced by enhanced bacterial productivity or bacterial growth in microgravity conditions, as is shown before in several other studies on bacteria in microgravity. Nonetheless, bacterial fuel cells have not been tested in space before. Currently foreseen applications are very promising for space flight and include waste disposal in manned space vehicles. This study describes a ‘space-first’test of bacterial fuel cells onboard the International Space Station using the Rhodoferax ferrireducens strain. We test if it is possible to use a bacterial fuel cell in 1g and under both simulated (RPM) and real microgravity conditions. Due to differences in magnitude of the output the data had to be normalized and cumulatively plotted. In all, it can be concluded that bacterial fuel cells show similar phases in the output under different gravitational conditions. Hence it can be concluded from a biological point of view that bacterial fuel cells do operate in space.