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.

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 in monolayer 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 in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. 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.

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.

Cancer is defined as a group of diseases characterized by abnormal cell growth, expansion, and progression with metastasis. Various signaling pathways are involved in its development. Malignant tumors exhibit a high morbidity and mortality. Cancer research increased our knowledge about some of the underlying mechanisms, but to this day, our understanding of this disease is unclear. High throughput omics technology and bioinformatics were successful in detecting some of the unknown cancer mechanisms. However, novel groundbreaking research and ideas are necessary. A stay in orbit causes biochemical and molecular biological changes in human cancer cells which are first, and above all, due to microgravity (µg). The µg-environment provides conditions that are not reachable on Earth, which allow researchers to focus on signaling pathways controlling cell growth and metastasis. Cancer research in space already demonstrated how cancer cell-exposure to µg influenced several biological processes being involved in cancer. This novel approach has the potential to fight cancer and to develop future cancer strategies. Space research has been shown to impact biological processes in cancer cells like proliferation, apoptosis, cell survival, adhesion, migration, the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors, among others. This concise review focuses on publications related to genetic, transcriptional, epigenetic, proteomic, and metabolomic studies on tumor cells exposed to real space conditions or to simulated µg using simulation devices. We discuss all omics studies investigating different tumor cell types from the brain and hematological system, sarcomas, as well as thyroid, prostate, breast, gynecologic, gastrointestinal, and lung cancers, in order to gain new and innovative ideas for understanding the basic biology of cancer.

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.

In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable low-mass model system, such as cell cultures, these devices simulating microgravity have been shown to produce results similar to those obtained in a space experiment under real microgravity conditions. One of these effects observed under real and simulated microgravity is the formation of spheroids from 2D adherent cancer cell cultures. Since real microgravity cannot be generated in a laboratory on Earth, we aimed to determine which forces lead to the detachment of individual FTC-133 thyroid cancer cells and the formation of tumor spheroids during culture with exposure to random positioning modes. To this end, we subdivided the RPM motion into different static and dynamic orientations of cell culture flasks. We focused on the molecular activation of the mechanosignaling pathways previously associated with spheroid formation in microgravity. Our results suggest that RPM-induced spheroid formation is a two-step process. First, the cells need to be detached, induced by the cell culture flask’s rotation and the subsequent fluid flow, as well as the presence of air bubbles. Once the cells are detached and in suspension, random positioning prevents sedimentation, allowing 3D aggregates to form. In a comparative shear stress experiment using defined fluid flow paradigms, transcriptional responses were triggered comparable to exposure of FTC-133 cells to the RPM. In summary, the RPM serves as a simulator of microgravity by randomizing the impact of Earth’s gravity vector especially for suspension (i.e., detached) cells. Simultaneously, it simulates physiological shear forces on the adherent cell layer. The RPM thus offers a unique combination of environmental conditions for in vitro cancer research.