Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.     

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.     

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.     

Finite Element Analysis of Osteocytes Mechanosensitivity Under Simulated Microgravity

It was found that the mechanosensitivity of osteocytes could be altered under simulated microgravity. However, how the mechanical stimuli as the biomechanical origins cause the bioresponse in osteocytes under microgravity is unclear yet. Computational studies may help us to explore the mechanical deformation changes of osteocytes under microgravity. Here in this paper, we intend to use the computational simulation to investigate the mechanical behavior of osteocytes under simulated microgravity. In order to obtain the shape information of osteocytes, the biological experiment was conducted under simulated microgravity prior to the numerical simulation The cells were rotated by a clinostat for 6 hours or 5 days and fixed, the cytoskeleton and the nucleus were immunofluorescence stained and scanned, and the cell shape and the fluorescent intensity were measured from fluorescent images to get the dimension information of osteocytes The 3D finite element (FE) cell models were then established based on the scanned image stacks. Several components such as the actin cortex, the cytoplasm, the nucleus, the cytoskeleton of F-actin and microtubules were considered in the model. The cell models in both 6 hours and 5 days groups were then imposed by three magnitudes (0.5, 10 and 15 Pa) of simulating fluid shear stress, with cell total displacement and the internal discrete components deformation calculated. The results showed that under the simulated microgravity: (1) the nuclear area and height statistically significantly increased, which made the ratio of membrane-cortex height to nucleus height statistically significantly decreased; (2) the fluid shear stress-induced maximum displacements and average displacements in the whole cell decreased, with the deformation decreasing amplitude was largest when exposed to 1.5Pa of fluid shear stress; (3) the fluid shear stress-induced deformation of cell membrane-cortex and cytoskeleton decreased, while the fluid shear stress-induced deformation of nucleus increased. The results suggested the mechanical behavior of whole osteocyte cell body was suppressed by simulated microgravity, and this decrement was enlarged with either the increasing amplitude of fluid shear stress or the duration of simulated microgravity. What’s more, the mechanical behavior of membrane-cortex and cytoskeleton was suppressed by the simulated microgravity, which indicated the mechanotransduction process in the cell body may be further inhibited. On the contrary, the cell nucleus deformation increased under simulated microgravity, which may be related to either the decreased amount of cytoskeleton or the increased volume occupied proportion of nucleus in whole cell under the simulated microgravity. The numerical results supported our previous biological experiments, and showed particularly affected cellular components under the simulated microgravity. The computational study here may help us to better understand the mechanism of mechanosensitivity changes in osteocytes under simulated microgravity, and further to explore the mechanism of the bone loss in space flight.     

Analysis of Statoliths Displacement in <Emphasis Type="Italic">Chara</Emphasis> Rhizoids for Validating the Microgravity-Simulation Quality of Clinorotation Modes

In single-celled rhizoids of the green algae Chara, positively gravitropic growth is governed by statoliths kept in a dynamically stable position 10–25 μ m above the cell tip by a complex interaction of gravity and actomyosin forces. Any deviation of the tube-like cells from the tip-downward orientation causes statoliths to sediment onto the gravisensitive subapical cell flank which initiates a gravitropic curvature response. Microgravity experiments have shown that abolishing the net tip-directed gravity force results in an actomyosin-mediated axial displacement of statoliths away from the cell tip. The present study was performed to critically assess the quality of microgravity simulation provided by different operational modes of a Random Positioning Machine (RPM) running with one axis (2D mode) or two axes (3D mode) and different rotational speeds (2D), speed ranges and directions (3D). The effects of 2D and 3D rotation were compared with data from experiments in real microgravity conditions (MAXUS sounding rocket missions). Rotational speeds in the range of 60–85 rpm in 2D and 3D modes resulted in a similar kinetics of statolith displacement as compared to real microgravity data, while slower clinorotation (2–11 rpm) caused a reduced axial displacement and a more dispersed arrangement of statoliths closer to the cell tip. Increasing the complexity of rotation by adding a second rotation axis in case of 3D clinorotation did not increase the quality of microgravity simulation, however, increased side effects such as the level of vibrations resulting in a more dispersed arrangement of statoliths. In conclusion, fast 2D clinorotation provides the most appropriate microgravity simulation for investigating the graviperception mechanism in Chara rhizoids, whereas slower clinorotation speeds and rotating samples around two axes do not improve the quality of microgravity simulation.     

Facilities for Simulation of Microgravity in the ESA Ground-Based Facility Programme

Knowledge of the role of gravity in fundamental biological processes and, consequently, the impact of exposure to microgravity conditions provide insight into the basics of the development of life as well as enabling long-term space exploration missions. However, experimentation in real microgravity is expensive and scarcely available; thus, a variety of platforms have been developed to provide, on Earth, an experimental condition comparable to real microgravity. With the aim of simulating microgravity conditions, different ground-based facilities (GBF) have been constructed such as clinostats and random positioning machines as well as magnets for magnetic levitation. Here, we give an overview of ground-based facilities for the simulation of microgravity which were used in the frame of an ESA ground-based research programme dedicated to providing scientists access to these experimental capabilities in order to prepare their space experiments.     

Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity

Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed.     

Actin dynamics in mouse fibroblasts in microgravity

After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.     

Topological considerations of parametric electromechanical devices and their parametric analysis

One of the methods for attaining free suspension of objects in magnetic fields is magnetic levitation by tuned circuits. Tuned-circuit levitators exhibit typical dynamic instability. However, the inherent tendency of the suspended object to oscillate may serve as a basis for the construction of relatively new types of machines. An attempt is made to treat, all these devices from a generalized point of view. A method similar to that used for the analysis of parametric electronic networks is suggested here to tackle parametric electromechanical systems. The paper concludes with two recent examples which illustrate how unexpected are the phenomena, where parametric electromechanical effects are of relevance. A peculiar mechanical instability which occurred in the VHF resonators used in particles accelerators, has been described recently. It has also been found that both a rotation of a suspended object as well as its levitation can be achieved by using a levitator with only one electromagnet.     

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.     

Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles

The actin cytoskeleton controls cell shape, motility, as well as intracellular molecular trafficking. The ability to remotely manipulate actin is therefore highly desirable as a tool to probe and manipulate biological processes at the molecular level. We demonstrate actin manipulation by labeling actin filaments with superparamagnetic iron oxide particles (IOPs) and applying a uniform magnetic field to affect actin orientation, polymerization and gliding on myosin. We show for the first time magnetic manipulation of magnetizable actin filaments at the molecular level while gliding on a bed of myosin molecules and during polymerization. A model for the magnetic alignment and guiding mechanism is proposed based on the torque from the induced molecular anisotropy due to interactions between neighboring IOPs distributed along magnetically labeled actin molecules.     

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     

Ions and water transmembrane transport in nervous and testicular cultured cells in low gravity conditions

Aim of the present study was to investigate on the possible alterations induced by on ground modeled microgravity on ion-water transport proteins at cellular level. For the purpose we used astrocytes, C₆ line, neurons (NT₂ line from human teratocarcinoma) and testicular cells (germ cells, Sertoli cells, and Leydig cells; primary cultures from trypsinised prepuberal pig testes). Modeled microgravity was achieved by a desktop 3D Random Positioning Machine, cultures were kept rotating for 30′, 1h and 24h. After 30′, immunopositivity for the antibodies to Na⁺/K⁺ATPase and Na⁺/K⁺/Cl^? co-transporters was greatly diminished, the plasma membrane appeared to be altered, and the mitochondria inner cristae were disrupted. Immunostaining to the antibody to the water channel aquaporin 4 was very bright. After 1h at random rotation immunostaining for the heat shock protein Hsp27 was visible, After 24h, immunostaining for the ion transport proteins was again like that of the controls, plasma membrane and the mitochondria were again normal. Immunostaining for aquaporin 4 become again similar to that of the controls. We conclude that low gravity induces only transient alterations in the cell’s transmembrane ion-water transport: the cells are able to adapt to the gravity vector changes in few hours.     

Characteristics of dynamic mass redistribution of epidermal growth factor receptor signaling in living cells measured with label-free optical biosensors

This paper reported the identification of a novel optical signature for epidermal growth factor (EGF) receptor signaling in human epidermoid carcinoma A431 cells mediated by EGF. The optical signature was based on dynamic mass redistribution (DMR) in living cells triggered by EGFR activation, as monitored in real time with resonant waveguide grating biosensors. Analysis of the modulation of the EGF-induced DMR signals by a variety of known modulators provided links of various targets to distinct steps in the cellular responses. Results showed that the dynamic mass redistribution in quiescent A431 cells mediated by EGF required EGFR tyrosine kinase activity, actin polymerization, and dynamin and mainly proceeded through MEK. The DMR signals obtained serve as integrated signatures for interaction networks in the EGFR signaling.     

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.     

用于晶体生长的地基无容器悬浮技术

空间微重力环境下几乎无对流和沉降,可为晶体生长提供一个相对稳定和均一的理想环境,易于得到尺寸较大的高质量单晶。但是,空间结晶实验成功率低,费用昂贵,实验机会受限。因此,研发各种空间微重力环境地基模拟技术具有重要意义。目前可用于晶体生长的地基无容器悬浮技术主要有空气动力悬浮、静电悬浮、电磁悬浮、液体界面悬浮、超声悬浮和磁场悬浮技术等。这些地基模拟技术可实现晶体的无容器悬浮生长,避免器壁对晶体生长的不良影响,提高晶体质量,为解决X射线单晶衍射技术中的瓶颈问题提供新途径,还可为在地基进行结晶动力学和机理研究提供简单易行的方法。从技术原理、优势、缺陷及在结晶(特别是蛋白质结晶)中的应用4个方面对这些技术逐一进行了介绍和评述。重点介绍了液体界面悬浮、超声悬浮和磁场悬浮技术这3种用于蛋白质晶体生长的较为成熟的地基无容器悬浮技术。     

Real-time and label-free detection of cellular response to signaling mediated by distinct subclasses of epidermal growth factor receptors

Epidermal growth factor receptors (EGFRs) have often shown two distinct binding affinities for epidermal growth factor. It is the high-affinity EGFR that is predominantly responsible for mediating the cell signaling that plays an indispensable role in cell growth, proliferation, motility, and differentiation. We applied the quartz crystal microbalance with dissipation monitoring (QCM-D) to track short-term cellular responses to EGFR signaling in human carcinoma A431 cells. Cellular responses to high- and low-affinity EGFR signaling were detected individually as well as simultaneously based on changes in mass and viscoelasticity of cells. These responses are associated with EGF-induced biological processes including the cytoskeleton remodeling and calcium influx. QCM-D provides a label-free sensor technology that can be exploited to investigate the role of high-affinity EGFR in cancer development and cancer prognosis.     

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.     

Simultaneous Quantification of the Interplay Between Molecular Turnover and Cell Mechanics by AFM–FRAP

Quantifying the adaptive mechanical behavior of living cells is essential for the understanding of their inner working and function. Yet, despite the establishment of quantitative methodologies correlating independent measurements of cell mechanics and its underlying molecular kinetics, explicit evidence and knowledge of the sensitivity of the feedback mechanisms of cells controlling their adaptive mechanics behavior remains elusive. Here, a combination of atomic force microscopy and fluorescence recovery after photobleaching is introduced offering simultaneous quantification and direct correlation of molecule kinetics and mechanics in living cells. Systematic application of this optomechanical atomic force microscopy–fluorescence recovery after photobleaching platform reveals changes in the actin turnover and filament lengths of ventral actin stress fibers in response to constant mechanical force at the apical actin cortex with a dynamic range from 0.1 to 10 nN, highlighting a direct relationship of active mechanosensation and adaptation of the cellular actin cytoskeleton. Simultaneous quantification of the relationship between molecule kinetics and cell mechanics may thus open‐up unprecedented insights into adaptive mechanobiological mechanisms of cells.     

微重力场下金属材料制备的发展现状

近年来微重力下制备金属材料的研究越来越引起人们的重视。简述了形成微重力的几种实验方法，综述了微重力下制备金属材料的发展现状。