Random Positioning Machine Research Articles

Protection of primary cilia is an effective countermeasure against the impairment of osteoblast function induced by simulated microgravity.

The molecular mechanism for the microgravity-induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)-simulated microgravity and that the microgravity-induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1μM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP-2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP-2, BMPRII, Smad1/5/8 and p-Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA-129-3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA-129-3p in ROBs significantly attenuated microgravity-induced inhibition of BMP-2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA-129-3p in microgravity-induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA-129-3p as an effective countermeasure against microgravity-induced loss of primary cilia and impairment of osteoblast function.

Simulated microgravity affects stroma-dependent ex vivo myelopoiesis

Microgravity is known negatively affect physiology of living beings, including hematopoiesis. Dysregulation of hematopoietic cells and supporting stroma relationships in bone marrow niche may be in charge. We compared the efficacy of ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) in presence of native or osteocommitted MSCs under simulated microgravity (Smg) using Random Positioning Machine (RPM). In comparison with 1 g, a decrease of MSC-associated HSPCs and an increase of floating HSPCs was observed after 7 days of Smg exposure. Among floating HSPCs, primitive progenitors were presented by late CD34+/133–. Total CFUs as well as erythroid (BFU-E) and granulocytic (CFU-G) numbers were lower. MSC-associated primitive HSPCs demonstrated increased proportion of late CD34+/133- in expense of early CD34-/133+. Osteo-MSCs preferentially supported late primitive CD34+ and more committed HSPCs as followed from increase of CFUs, and CD235a+ erythroid progenitors. Under Smg, an increased VEGF, eotaxin, and GRO-a levels, and a decrease in RANTES were found in the osteo-MSC-HSPC co-cultures. IL-6,-8, -13, G-CSF, GRO-a, MCP-3, MIP-1b, VEGF increased in co-culture with osteo-MSCs vs intact MSCs. Based on the findings, the misbalance between primitive/committed HSPCs and a decrease in hematopoiesis-supportive activity of osteocommitted cells are supposed to underline hematopoietic disorders during space flights.

Simulated Microgravity Created Using a Random Positioning Machine Induces Changes in the Physiology of the Fusarium solani Species Complex.

Fusarium is a phytopathogenic fungus involved in human pathology and is present in space stations. It is essential to understand the effects of microgravity on the physiology of this fungus to determine the potential risks to the health of crew members and to propose the necessary countermeasures. This study aimed to determine changes in the physiological parameters of the Fusarium solani species complex under simulated microgravity generated using a random positioning machine (RPM) and phenotypic approaches. We observed increased growth, spore production, and germination while biofilm production was reduced under RPM exposure. These in vitro data show the importance of further studying this fungus as it has been repeatedly demonstrated that microgravity weakens the immune system of astronauts.

A New Random Positioning Machine Modification Applied for Microgravity Simulation in Laboratory Experiments with Rats

The study presents a newly constructed modification of a random positioning machine (RPM) used in 3D-clinostat and in random mode. The main purpose is to provide an RPM animal model that uses up to four experimental animals simultaneously. In order to validate our RPM, the gravity dispersion and its magnitude are compared with the ones of a traditional machine. The results showed no crucial deviations in gravity dispersion and its time-averaged value in all sets of parameters. Furthermore, a posteriori stress tests are conducted on three Wistar male rats groups in order to estimate the level of stress from the setup. The social trait results suggest that the group exposed to our device has no increase in anxiety.

Cell Responses to Simulated Microgravity and Hydrodynamic Stress Can Be Distinguished by Comparative Transcriptomics

The human immune system is compromised in microgravity (MG) conditions during an orbital flight and upon return to Earth. T cells are critical for the immune response and execute their functions via actin-mediated immune cell-cell interactions that could be disturbed by MG conditions. In our study, we have applied two conventional platforms to simulate MG conditions: fast rotating clinostat (CL) and random positioning machine (RPM), followed by global T cell transcriptome analysis using RNA sequencing. Noteworthily, both selected rotational simulated MG platforms employ forced cell movement in cultural medium and expose cells to shear forces, therefore inducing certain cell response to hydrodynamic stress. We demonstrate that the T cell transcriptome profile in response to simulated MG treatment was clearly distinguishable from the T cell transcriptome response to hydrodynamic stress (HS). Gene expression profiling of genes related to or involved in actin cytoskeleton networks using RT-qPCR confirmed two sets of differentially regulated genes in the T cell response to MG or to HS. Several key genes potentially involved in T cell gravisensing (Fam163b, Dnph1, Trim34, Upk-1b) were identified. A number of candidate biomarker genes of the response to MG (VAV1, VAV2, VAV3, and NFATC2) and of the response to HS (ITGAL, ITGB1, ITGB2, RAC1, and RAC2) could be used to distinguish between these processes on the gene transcription level. Together, MG induces changes in the overall transcriptome of T cells, leading to specific shifts in the expression of cytoskeletal network genes.

Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

The multifaceted adverse effects of reduced gravity pose a significant challenge to human spaceflight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts’ function are less well understood. Utilizing the software-driven newer version of the Random Positioning Machine (RPMSW), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon (0.16 G), and microgravity (Micro, ~10−3 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration.

Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

Effects of Titanium Dioxide Nanoparticles on Cell Growth and Migration of A549 Cells under Simulated Microgravity

With the increasing application of nanomaterials in aerospace technology, the long-term space exposure to nanomaterials especially in the space full of radiation coupled with microgravity condition has aroused great health concerns of the astronauts. However, few studies have been conducted to assess these effects, which are crucial for seeking the possible intervention strategy. Herein, using a random positioning machine (RPM) to simulate microgravity, we investigated the behaviors of cells under simulated microgravity and also evaluated the possible toxicity of titanium dioxide nanoparticles (TiO2 NPs), a multifunctional nanomaterial with potential application in aerospace. Pulmonary epithelial cells A549 were exposed to normal gravity (1 g) and simulated gravity (~10−3 g), respectively. The results showed that simulated microgravity had no significant effect on the viability of A549 cells as compared with normal gravity within 48 h. The effects of TiO2 NPs exposure on cell viability and apoptosis were marginal with only a slightly decrease in cell viability and a subtle increase in apoptosis rate observed at a high concentration of TiO2 NPs (100 μg/mL). However, it was observed that the exposure to simulated microgravity could obviously reduce A549 cell migration compared with normal gravity. The disruption of F-actin network and the deactivation of FAK (Tyr397) might be responsible for the impaired mobility of simulated microgravity-exposed A549 cells. TiO2 NPs exposure inhibited cell migration under two different gravity conditions, but to different degrees, with a milder inhibition under simulated microgravity. Meanwhile, it was found that A549 cells internalized more TiO2 NPs under normal gravity than simulated microgravity, which may account for the lower cytotoxicity and the lighter inhibition of cell migration induced by the same exposure concentration of TiO2 NPs under simulated microgravity at least partially. Our study has provided some tentative information on the effects of TiO2 NPs exposure on cell behaviors under simulated microgravity.

Simulated microgravity promotes oxidative stress-induced apoptosis in ARPE-19 cells associated with Nrf2 signaling pathway

Spaceflight associated neuro-ocular syndrome (SANS) has become a high-risk complication for astronauts in the microgravity environment. Growing evidences suggest that altered hemodynamics of the cerebrospinal fluid system under microgravity may affect optic nerve-related structures and lead to ocular diseases. Retinal pigment epithelial (RPE) cells play a pivotal role in maintaining the blood-retinal barrier, participating in the metabolism of visual circulation. Previous study has observed that the structure, shape, adhesion, migration and angiogenesis of RPE cells were significantly changed under microgravity. However, the interrelated molecular mechanisms underlying the cytological functions of RPE cells under microgravity have yet to be elucidated. Here, we exposed ARPE-19 cells in Random Positioning Machine (RPM) system. The preliminary analysis of cytological functions found that the viability of ARPE-19 cells was decreased and the cell cycle was arrested in S phase. Further results revealed that elevated ROS in microgravity caused oxidative stress, resulting in the activation of the Nrf2-HO-1 pathway. Meanwhile, cells subjected to oxidative damage result in apoptosis by regulating the expression of apoptosis-related genes. Taken together, these findings have significant implications for the understanding the molecular mechanism of SANS.

Exposure to Random Positioning Machine Alters the Mineralization Process and PTX3 Expression in the SAOS-2 Cell Line.

Bone loss is among the most frequent changes seen in astronauts during space missions. Although weightlessness is known to cause high bone resorption and a rapid decrease in bone minerals and calcium, the underlying mechanisms are not yet fully understood. In our work, we investigated the influence of random positioning machine (RPM) exposure on the mineralization process in the SAOS-2 cell line, in osteogenic and non-osteogenic conditions, by examining changes in their mineralizing capacity and in the expression of PTX3, a positive regulator of bone mineralization. We analyzed cell viability by MTS assay and the mineralization process after staining with Toluidine Blue and Alizarin Red, while PTX3 expression was investigated by immunocytochemistry and western blotting analysis. Our results showed that RPM exposure increased cells’ viability and improved their mineralizing competence when not treated with osteogenic cocktail. In contrast, in osteogenic conditions, cells exposed to RPM showed a reduction in the presence of calcification-like structures, mineral deposits and PTX3 expression, suggesting that the effects of RPM exposure on mineralizing matrix deposition depend on the presence of osteogenic factors in the culture medium. Further studies will be needed to clarify the role of potential mineralization markers in the cellular response to the simulated biological effects of microgravity, paving the way for a new approach to treating osteoporosis in astronauts exposed to spaceflight.

Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis

BackgroundThe impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed.MethodsExploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days.ResultsOur results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair β-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation.ConclusionsSMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology.

Three-Dimensional Growth of Prostate Cancer Cells Exposed to Simulated Microgravity.

Prostate cancer metastasis has an enormous impact on the mortality of cancer patients. Factors involved in cancer progression and metastasis are known to be key players in microgravity (µg)-driven three-dimensional (3D) cancer spheroid formation. We investigated PC-3 prostate cancer cells for 30 min, 2, 4 and 24 h on the random positioning machine (RPM), a device simulating µg on Earth. After a 24 h RPM-exposure, the cells could be divided into two groups: one grew as 3D multicellular spheroids (MCS), the other one as adherent monolayer (AD). No signs of apoptosis were visible. Among others, we focused on cytokines involved in the events of metastasis and MCS formation. After 24 h of exposure, in the MCS group we measured an increase in ACTB, MSN, COL1A1, LAMA3, FN1, TIMP1, FLT1, EGFR1, IL1A, IL6, CXCL8, and HIF1A mRNA expression, and in the AD group an elevation of LAMA3, COL1A1, FN1, MMP9, VEGFA, IL6, and CXCL8 mRNAs compared to samples subjected to 1 g conditions. Significant downregulations in AD cells were detected in the mRNA levels of TUBB, KRT8, IL1B, IL7, PIK3CB, AKT1 and MTOR after 24 h. The release of collagen-1α1 and fibronectin protein in the supernatant was decreased, whereas the secretion of IL-6 was elevated in 24 h RPM samples. The secretion of IL-1α, IL-1β, IL-7, IL-2, IL-8, IL-17, TNF-α, laminin, MMP-2, TIMP-1, osteopontin and EGF was not significantly altered after 24 h compared to 1 g conditions. The release of soluble factors was significantly reduced after 2 h (IL-1α, IL-2, IL-7, IL-8, IL-17, TNF-α, collagen-1α1, MMP-2, osteopontin) and elevated after 4 h (IL-1β, IL-2, IL-6, IL-7, IL-8, TNF-α, laminin) in RPM samples. Taken together, simulated µg induced 3D growth of PC-3 cancer cells combined with a differential expression of the cytokines IL-1α, IL-1β, IL-6 and IL-8, supporting their involvement in growth and progression of prostate cancer cells.

Microgravity Modifies the Phenotype of Fibroblast and Promotes Remodeling of the Fibroblast-Keratinocyte Interaction in a 3D Co-Culture Model.

Microgravity impairs tissue organization and critical pathways involved in the cell–microenvironment interplay, where fibroblasts have a critical role. We exposed dermal fibroblasts to simulated microgravity by means of a Random Positioning Machine (RPM), a device that reproduces conditions of weightlessness. Molecular and structural changes were analyzed and compared to control samples growing in a normal gravity field. Simulated microgravity impairs fibroblast conversion into myofibroblast and inhibits their migratory properties. Consequently, the normal interplay between fibroblasts and keratinocytes were remarkably altered in 3D co-culture experiments, giving rise to several ultra-structural abnormalities. Such phenotypic changes are associated with down-regulation of α-SMA that translocate in the nucleoplasm, altogether with the concomitant modification of the actin-vinculin apparatus. Noticeably, the stress associated with weightlessness induced oxidative damage, which seemed to concur with such modifications. These findings disclose new opportunities to establish antioxidant strategies that counteract the microgravity-induced disruptive effects on fibroblasts and tissue organization.

Role of Myostatin in Muscle Degeneration by Random Positioning Machine Exposure: An in vitro Study for the Treatment of Sarcopenia.

Several scientific evidence have shown that exposure to microgravity has a significant impact on the health of the musculoskeletal system by altering the expression of proteins and molecules involved in bone–muscle crosstalk, which is also observed in the research of microgravity effect simulation. Among these, the expression pattern of myostatin appears to play a key role in both load-free muscle damage and the progression of age-related musculoskeletal disorders, such as osteoporosis and sarcopenia. Based on this evidence, we here investigated the efficacy of treatment with anti-myostatin (anti-MSTN) antibodies on primary cultures of human satellite cells exposed to 72 h of random positioning machine (RPM). Cell cultures were obtained from muscle biopsies taken from a total of 30 patients (controls, osteoarthritic, and osteoporotic) during hip arthroplasty. The Pax7 expression by immunofluorescence was carried out for the characterization of satellite cells. We then performed morphological evaluation by light microscopy and immunocytochemical analysis to assess myostatin expression. Our results showed that prolonged RPM exposure not only caused satellite cell death, but also induced changes in myostatin expression levels with group-dependent variations. Surprisingly, we observed that the use of anti-MSTN antibodies induced a significant increase in cell survival after RPM exposure under all experimental conditions. Noteworthy, we found that the negative effect of RPM exposure was counteracted by treatment with anti-MSTN antibodies, which allowed the formation of numerous myotubes. Our results highlight the role of myostatin as a major effector of the cellular degeneration observed with RPM exposure, suggesting it as a potential therapeutic target to slow the muscle mass loss that occurs in the absence of loading.

Simulated Microgravity Potentiates Hematopoietic Differentiation of Human Pluripotent Stem Cells and Supports Formation of 3D Hematopoietic Cluster.

Microgravity has been shown to induces many changes in proliferation, differentiation and growth behavior of stem cells. Little is known about the effect of microgravity on hematopoietic differentiation of pluripotent stem cells (PSCs). In this study, we used the random position machine (RPM) to investigate whether simulated microgravity (SMG) allows the induction of hematopoietic stem/progenitor cell (HSPC) derived from human embryonic stem cells (hESCs) in vitro. The results showed that SMG facilitates hESCs differentiate to HSPC with more efficient induction of CD34+CD31+ hemogenic endothelium progenitors (HEPs) on day 4 and CD34+CD43+ HSPC on day 7, and these cells shows an increased generation of functional hematopoietic cells in colony-forming unit assay when compared with normal gravity (NG) conditions. Additionally, we found that SMG significantly increased the total number of cells on day 4 and day 7 which formed more 3D cell clusters. Transcriptome analysis of cells identified thousands of differentially expressed genes (DEGs) between NG and SMG. DEGs down-regulated were enriched in the axonogenesis, positive regulation of cell adhesion, cell adhesion molecule and axon guidance, while SMG resulted in the up-regulation of genes were functionally associated with DNA replication, cell cycle, PI3K-Akt signaling pathway and tumorigenesis. Interestingly, some key gene terms were enriched in SMG, like hypoxia and ECM receptor interaction. Moreover, HSPC obtained from SMG culture conditions had a robust ability of proliferation in vitro. The proliferated cells also had the ability to form erythroid, granulocyte and monocyte/macrophage colonies, and can be induced to generate macrophages and megakaryocytes. In summary, our data has shown a potent impact of microgravity on hematopoietic differentiation of hPSCs for the first time and reveals an underlying mechanism for the effect of SMG on hematopoiesis development.

ВЛИЯНИЕ МОДЕЛИРУЕМОЙ МИКРОГРАВИТАЦИИ НА СОДЕРЖАНИЕ мРНК ГЕНОВ, КОДИРУЮЩИХ ЦИТОСКЕЛЕТНЫЕ БЕЛКИ, И АЦЕТИЛИРОВАНИЕ ГИСТОНОВ В ЯИЧНИКАХ DROSOPHILA MELANOGASTER

Maintenance of the human reproductive health is vital in context of preserving living species in remote space missions. Changes in the gene expression due to microgravity can lead to degradation of fertility; however, the mechanism that triggers these changes is not fully understood. In our investigation we determined mRNA levels in the prime cytoskeletal proteins and analyzed histone methylation and acetylation activities in Drosophila melanogaster ovaries during 79-hr microgravity modeled with the use of a random positioning machine (RPM), and per os introduction of essential phospholipids. The results showed that the fruit flies exposure to modeled microgravity over the entire oogenesis cycle increased levels of mRNA genes encoding the cytoskeletal proteins in oocytes and that this can be, probably, associated with increased acetylation of the histone H3 Lys9 and Lys27. Essential phospholipids seem to prevent growth and/or initiate mRNA reductions in the majority of cytoskeleton elements in the flies exposed to modeled microgravity; however, the tubulin and lamin B binding proteins increased in the respective controls. Essential phospholipids leveled histone 3 Lys9 acetylation in modeled microgravity but enhanced Lys27 acetylation in the control.

Nutraceuticals Synergistically Promote Osteogenesis in Cultured 7F2 Osteoblasts and Mitigate Inhibition of Differentiation and Maturation in Simulated Microgravity.

Microgravity is known to impact bone health, similar to mechanical unloading on Earth. In the absence of countermeasures, bone formation and mineral deposition are strongly inhibited in Space. There is an unmet need to identify nutritional countermeasures. Curcumin and carnosic acid are phytonutrients with anticancer, anti-inflammatory, and antioxidative effects and may exhibit osteogenic properties. Zinc is a trace element essential for bone formation. We hypothesized that these nutraceuticals could counteract the microgravity-induced inhibition of osteogenic differentiation and function. To test this hypothesis, we cultured 7F2 murine osteoblasts in simulated microgravity (SMG) in a Random Positioning Machine in the presence and absence of curcumin, carnosic acid, and zinc and evaluated cell proliferation, function, and differentiation. SMG enhanced cell proliferation in osteogenic medium. The nutraceuticals partially reversed the inhibitory effects of SMG on alkaline phosphatase (ALP) activity and did not alter the SMG-induced reduction in the expression of osteogenic marker genes in osteogenic medium, while they promoted osteoblast proliferation and ALP activity in the absence of traditional osteogenic media. We further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.

Morphofunctional, viability and antioxidant system alterations on rat primary testicular cells exposed to simulated microgravity

This study focused on effects induced by Short-term Simulated Microgravity (SMG) condition on primary cell culture from pre-pubertal Wistar rats testis. Cells were analyzed for cytoskeletal and Sex Hormone Binding Globulin (SHBG/ABP) changes by immunofluorescence technique, for antioxidant system exploiting RT-PCR and cell viability. Cells were cultured for 6 and 24h on a three-dimensional clinostat, Random Positioning Machine (RPM). At the end of each experiment, once stopped the RPM rotation, cells were either fixed in paraformaldehyde or lysed and RNA extracted. In cells exposed to SMG the cytoskeleton became disorganized, microtubules fragmented and SHBG was already undetectable after 6h of treatment. Moreover, various antioxidant systems significantly increased after 24h of SMG exposure. Initially, SMG seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Studies on changes induced by short-term altered gravity conditions, carried out in real microgravity, could give more information on steroidogenesis and germ cell differentiation within the testis exposed to this condition and confirm the validity of simulation approach.

Biological and Mechanical Characterization of the Random Positioning Machine (RPM) for Microgravity Simulations.

The rapid improvement of space technologies is leading to the continuous increase of space missions that will soon bring humans back to the Moon and, in the coming future, toward longer interplanetary missions such as the one to Mars. The idea of living in space is charming and fascinating; however, the space environment is a harsh place to host human life and exposes the crew to many physical challenges. The absence of gravity experienced in space affects many aspects of human biology and can be reproduced in vitro with the help of microgravity simulators. Simulated microgravity (s-μg) is applied in many fields of research, ranging from cell biology to physics, including cancer biology. In our study, we aimed to characterize, at the biological and mechanical level, a Random Positioning Machine in order to simulate microgravity in an in vitro model of Triple-Negative Breast Cancer (TNBC). We investigated the effects played by s-μg by analyzing the change of expression of some genes that drive proliferation, survival, cell death, cancer stemness, and metastasis in the human MDA-MB-231 cell line. Besides the mechanical verification of the RPM used in our studies, our biological findings highlighted the impact of s-μg and its putative involvement in cancer progression.

Simulating microgravity using a random positioning machine for inducing cellular responses to mechanotransduction in human osteoblasts.

The mechanotransduction pathways that mediate cellular responses to contact forces are better understood than those that mediate response to distance forces, especially the force of gravity. Removing or reducing gravity for significant periods of time involves either sending samples to space, inducing diamagnetic levitation with high magnetic fields, or continually reorienting samples for a period, all in a manner that supports cell culturing. Undesired secondary effects due to high magnetic fields or shear forces associated with fluid flow while reorienting must be considered in the designof ground-based devices. We have developed a lab-friendly and compact random positioning machine (RPM) that fits in a standard tissue culture incubator. Using a two-axis gimbal, it continually reorients samples in a manner that produces an equal likelihood that all possible orientations are visited. We contribute a new control algorithm by which the distribution of probabilities over all possible orientations is completely uniform. Rather than randomly varying gimbal axis speed and/or direction as in previous algorithms (which produces non-uniform probability distributions of orientation), we use inverse kinematics to follow a trajectory with a probability distribution of orientations that is uniform by construction. Over a time period of 6 h of operation using our RPM, the average gravity is within 0.001 23% of the gravity of Earth. Shear forces are minimized by limiting the angular speed of both gimbal motors to under 42 °/s. We demonstrate the utility of our RPM by investigating the effects of simulated microgravity on adherent human osteoblasts immediately after retrieving samples from our RPM. Cytoskeletal disruption and cell shape changes were observed relative to samples cultured in a 1 g environment. We also found that subjecting human osteoblasts in suspension to simulated microgravity resulted in less filamentous actin and lower cell stiffness.