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Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight., + ID der Publikation: hslu_88043 + Art des Beitrages: Wissenschaftliche Medien + Jahrgang: 2022 + Sprache: Englisch + Letzte Aktualisierung: 2022-01-25 16:01:32

We introduce a new benchtop microgravity simulator (MGS) that is scalable and easy to use. Its working principle is similar to that of random positioning machines (RPM), commonly used in research laboratories and regarded as one of the gold standards for simulating microgravity. The improvement of the MGS concerns mainly the algorithms controlling the movements of the samples and the design that, for the first time, guarantees equal treatment of all the culture flasks undergoing simulated microgravity. Qualification and validation tests of the new device were conducted with human bone marrow stem cells (bMSC) and mouse skeletal muscle myoblasts (C2C12). bMSC were cultured for 4 days on the MGS and the RPM in parallel. In the presence of osteogenic medium, an overexpression of osteogenic markers was detected in the samples from both devices. Similarly, C2C12 cells were maintained for 4 days on the MGS and the rotating wall vessel (RWV) device, another widely used microgravity simulator. Significant downregulation of myogenesis markers was observed in gravitationally unloaded cells. Therefore, similar results can be obtained regardless of the used simulated microgravity devices, namely MGS, RPM, or RWV. The newly developed MGS device thus offers easy and reliable long-term cell culture possibilities under simulated microgravity conditions. Currently, upgrades are in progress to allow real-time monitoring of the culture media and liquids exchange while running. This is of particular interest for long-term cultivation, needed for tissue engineering applications. Tissue grown under real or simulated microgravity has specific features, such as growth in three-dimensions (3D). Growth in weightlessness conditions fosters mechanical, structural, and chemical interactions between cells and the extracellular matrix in any direction.

Adequate mechanical stimulation is essential for cellular health and tissue maintenance, including articular cartilage, which lines the articulating bones in joints. Chondrocytes, which are the sole cells found in articular cartilage, are responsible for matrix synthesis, maintenance and degradation. It is generally believed that chondrocytes require mechanical stimuli through daily physical activity for adequate cartilage homeostasis. However, to date, the molecular mechanisms of cellular force sensing (mechanotransduction) are not fully understood. Among other mechanisms, the cytoskeleton is thought to play a key role. Despite that gravity is a very small force at the cellular level, cytoskeletal adaptations have been observed under altered gravity conditions of a parabolic flight in multiple cell types. In this study, we developed a novel hardware which allowed to chemically fix primary bovine chondrocytes at 7 time points over the course of a 31-parabola flight. The samples were subsequently stained for the microtubules and vimentin network and microscopic images were acquired. The images showed a large heterogeneity among the cells in morphology as well as in the structure of both networks. In all, no changes or adaptions in cytoskeleton structure could be detected over the course of the parabolic flight., + ID der Publikation: hslu_78813 + Art des Beitrages: Wissenschaftliche Medien + Jahrgang: 2020 + Sprache: Englisch + Letzte Aktualisierung: 2020-09-08 15:15:01

Mechanical unloading by microgravity (or weightlessness) conditions triggers profound adaptation processes at the cellular and organ levels. Among other mechanisms, mechanosensitive ion channels are thought to play a key role in allowing cells to transduce mechanical forces. Previous experiments performed under microgravity have shown that gravity affects the gating properties of ion channels. Here, a method is described to record a calcium-dependent current in native Xenopus laevis oocytes under microgravity conditions during a parabolic flight. A 3-voltage-step protocol was applied to provoke a calcium-dependent current. This current increased with extracellular calcium concentration and could be reduced by applying extracellular gadolinium. The custom-made “OoClamp” hardware was validated by comparing the results of the 3-voltage-step protocol to results obtained with a well-established two-electrode voltage clamp (TEVC). In the context of the 2nd Swiss Parabolic Flight Campaign, we tested the OoClamp and the method. The setup and experiment protocol worked well in parabolic flight. A tendency that the calcium-dependent current was smaller under microgravity than under 1 g condition could be observed. However, a conclusive statement was not possible due to the small size of the data base that could be gathered.

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.

Advances in electrophysiological experiments have led to the discovery of mechanosensitive ion channels (MSCs) and the identification of the physiological function of specific MSCs. They are believed to play important roles in mechanosensitive pathways by allowing for cells to sense their mechanical environment. However, the physiological function of many MSCs has not been conclusively identified. Therefore, experiments have been developed that expose cells to various mechanical loads, such as shear flow, membrane indentation, osmotic challenges and hydrostatic pressure. In line with these experiments, mechanical unloading, as experienced in microgravity, represents an interesting alternative condition, since exposure to microgravity leads to a series of physiological adaption processes. As outlined in this review, electrophysiological experiments performed in microgravity have shown an influence of gravity on biological functions depending on ion channels at all hierarchical levels, from the cellular level to organs. In this context, calcium signaling represents an interesting cellular pathway, as it involves the direct action of calcium-permeable ion channels, and specific gravitatic cells have linked graviperception to this pathway. Multiple key proteins in the graviperception pathways have been identified. However, measurements on vertebrae cells have revealed controversial results. In conclusion, electrophysiological experiments in microgravity have shown that ion-channel-dependent physiological processes are altered in mechanically unloaded conditions. Future experiments may provide a better understanding of the underlying mechanisms.

Random Positioning Machines (RPMs) are widely used as tools to simulate microgravity on ground. They consist of two gimbal mounted frames, which constantly rotate biological samples around two perpendicular axes and thus distribute the Earth's gravity vector in all directions over time. In recent years, the RPM is increasingly becoming appreciated as a laboratory instrument also in non-space-related research. For instance, it can be applied for the formation of scaffold-free spheroid cell clusters. The kinematic rotation of the RPM, however, does not only distribute the gravity vector in such a way that it averages to zero, but it also introduces local forces to the cell culture. These forces can be described by rigid body analysis. Although RPMs are commonly used in laboratories, the fluid motion in the cell culture flasks on the RPM and the possible effects of such on cells have not been examined until today; thus, such aspects have been widely neglected. In this study, we used a numerical approach to describe the fluid dynamic characteristic occurring inside a cell culture flask turning on an operating RPM. The simulations showed that the fluid motion within the cell culture flask never reached a steady state or neared a steady state condition. The fluid velocity depends on the rotational velocity of the RPM and is in the order of a few centimeters per second. The highest shear stresses are found along the flask walls; depending of the rotational velocity, they can reach up to a few 100 mPa. The shear stresses in the "bulk volume," however, are always smaller, and their magnitude is in the order of 10 mPa. In conclusion, RPMs are highly appreciated as reliable tools in microgravity research. They have even started to become useful instruments in new research fields of mechanobiology. Depending on the experiment, the fluid dynamic on the RPM cannot be neglected and needs to be taken into consideration. The results presented in this study elucidate the fluid motion and provide insight into the convection and shear stresses that occur inside a cell culture flask during RPM experiments., + ID der Publikation: hslu_50957 + Art des Beitrages: Wissenschaftliche Medien + Sprache: Englisch + Letzte Aktualisierung: 2018-02-28 09:20:38

It is not fully understood how cells detect external mechanical forces, but mechanosensitive ion channels play important roles in detecting and translating physical forces into biological responses (mechanotrans-duction). With the “OoClamp” device, we developed a tool to study electrophysiological processes, including the gating properties of ion channels under various gravity conditions. The “OoClamp” device uses an adapted patch clamp technique and is operational during parabolic flight and centrifugation up to 20 g. In the framework of the REXUS/BEXUS program, we have further developed the “OoClamp” device with the goal of conducting electrophysiological experiments aboard a flying sounding rocket., + ID der Publikation: hslu_51260 + Art des Beitrages: Wissenschaftliche Medien + Sprache: Englisch + Letzte Aktualisierung: 2018-02-09 11:08:15

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.

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype inmonolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days instatic monolayer culture; (b) two days in static monolayer culture, followed by six days of RPMexposure; and, (c) eight days of RPMexposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions.The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

Random Positioning Machines (RPMs) have been used since many years as a ground-based model to simulate microgravity. In this review we discuss several aspects of the RPM. Recent technological development has expanded the operative range of the RPM substantially. New possibilities of live cell imaging and partial gravity simulations, for example, are of particular interest. For obtaining valuable and reliable results from RPM experiments, the appropriate use of the RPM is of utmost importance. The simulation of microgravity requires that the RPM’s rotation is faster than the biological process under study, but not so fast that undesired side effects appear. It remains a legitimate question, however, whether the RPM can accurately and reliably simulate microgravity conditions comparable to real microgravity in space. We attempt to answer this question by mathematically analyzing the forces working on the samples while they are mounted on the operating RPM and by comparing data obtained under real microgravity in space and simulated microgravity on the RPM. In conclusion and after taking the mentioned constraints into consideration, we are convinced that simulated microgravity experiments on the RPM are a valid alternative for conducting examinations on the influence of the force of gravity in a fast and straightforward approach., + ID der Publikation: hslu_29003 + Art des Beitrages: Wissenschaftliche Medien + Sprache: Englisch + Letzte Aktualisierung: 2018-02-09 10:46:56

Important in biotechnology is the establishment of cell culture methods that reflect the in vivo situation accurately. One approach for reaching this goal is through 3D cell cultivation that mimics tissue or organ structures and functions. We present here a newly designed and constructed random positioning incubator (RPI) that enables 3D cell culture in simulated microgravity (0 g). In addition to growing cells in a weightlessness-like environment, our RPI enables long-duration cell cultivation under various gravitational loads, ranging from close to 0 g to almost 1 g. This allows the study of the mechanotransductional process of cells involved in the conversion of physical forces to an appropriate biochemical response. Gravity is a type of physical force with profound developmental implications in cellular systems as it modulates the resulting signaling cascades as a consequence of mechanical loading. The experiments presented here were conducted on mouse skeletal myoblasts and human lymphocytes, two types of cells that have been shown in the past to be particularly sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are conducted. The scientific data gathered may not only improve the sustainment of human life in space, but also lead to the design of alternative countermeasures against diseases related to impaired mechanosensation and downstream signaling processes on earth., Biotechnology and Bioengineering, 111 (6), ISSN:0006-3592, ISSN:1097-0290