Search Results (19)

Microgravity (µg)-generated three-dimensional (3D) multicellular aggregates can serve as models of tissue and disease development. They are relevant in the fields of cancer and in vitro metastasis or regenerative medicine (tissue engineering). Driven by the 3R concept—replacement, reduction, and refinement of animal testing—µg-exposure of human cells represents a new alternative method that avoids animal experiments entirely. New Approach Methodologies (NAMs) are used in biomedical research, pharmacology, toxicology, cancer research, radiotherapy, and translational regenerative medicine. Various types of human cells grow as 3D spheroids or organoids when exposed to µg-conditions provided by µg simulating instruments on Earth. Examples for such µg-simulators are the Rotating Wall Vessel, the Random Positioning Machine, and the 2D or 3D clinostat. This review summarizes the most recent literature focusing on µg-engineered tissues. We are discussing all reports examining different tumor cell types from breast, lung, thyroid, prostate, and gastrointestinal cancers. Moreover, we are focusing on µg-generated spheroids and organoids derived from healthy cells like chondrocytes, stem cells, bone cells, endothelial cells, and cardiovascular cells. The obtained data from NAMs and µg-experiments clearly imply that they can support translational medicine on Earth. Full article

Spaceflight induces a wide array of effects on the human body, notably including pathological changes mediated by alterations in gravity. Abnormalities in the formation of primary cilia (ciliogenesis) can lead to cell cycle arrest and decreased epithelial cell proliferation, thereby delaying wound healing. To investigate the effect of microgravity on ciliogenesis in bronchial epithelial cells, we used a 3D clinostat to generate simulated microgravity (SMG) conditions. When BEAS-2B bronchial epithelial cells were exposed to SMG for 72 h, their proliferation was significantly reduced. The expression of Ki-67, which is not expressed in the G0 phase, decreased under SMG. Conversely, the expression of p27, which is expressed in the G0 and G1 phases, increased under SMG. These results suggest that SMG led to an increase in the number of cells in the quiescent phase. When the mRNA expressions of ARL13B (a marker of cilia assembly) and disassembly-related genes (Aurora A, NDE1, HDAC6, and DVL2) were evaluated, SMG upregulated ciliary assembly markers and downregulated disassembly markers. In addition, SMG increased the cilia length and number of ciliated cells. These findings suggest that SMG contributes to reduced cell proliferation through cell cycle arrest by disrupting normal ciliogenesis. Our findings indicate that SMG could delay lung injury by decreasing cell proliferation. Full article

Space flight has many adverse effects on the physiological functions of astronauts. Certain similarities have been observed in some physiological processes of rodents and astronauts in space, although there are also differences. These similarities make rodents helpful models for initial investigations into space-induced physiological changes. This study uses a 3D-Clinostat to simulate microgravity and explores the role of microgravity in space flight-induced liver and brain abnormalities by comparing changes in the gut microbiota, serum metabolites, and the function and physiological biochemistry of liver and brain tissues between the simulated microgravity (SMG) group mice and the wild type (WT) group mice. The study, based on hematoxylin-eosin (HE) staining, 16S sequencing technology, and non-targeted metabolomics analysis, shows that the gut tissue morphology of the SMG group mice is abnormal, and the structure of the gut microbiota and the serum metabolite profile are imbalanced. Furthermore, using PICRUST 2 technology, we have predicted the functions of the gut microbiota and serum metabolites, and the results indicate that the liver metabolism and functions (including lipid metabolism, amino acid metabolism, and sugar metabolism, etc.) of the SMG group mice are disrupted, and the brain tissue metabolism and functions (including neurotransmitters and hormone secretion, etc.) are abnormal, suggesting a close relationship between microgravity and liver metabolic dysfunction and brain dysfunction. Additionally, the high similarity in the structure of the gut microbiota and serum metabolite profile between the fecal microbiota transplant (FMT) group mice and the SMG group mice, and the physiological and biochemical differences in liver and brain tissues compared to the WT group mice, suggest that microgravity induces imbalances in the gut microbiota, which in turn triggers abnormalities in liver and brain metabolism and function. Finally, through MetaMapp analysis and Pearson correlation analysis, we found that valeric acid, a metabolite of gut microbiota, is more likely to be the key metabolite that relates to microgravity-induced gut microbiota abnormalities, disorders of amino acid and lipid metabolism, and further induced metabolic or functional disorders in the liver and brain. This study has significant practical application value for deepening the understanding of the adaptability of living organisms in the space environment. Full article

Research into the mechanisms by which gravity influences spermatozoa has implications for maintaining the species in deep space exploration and may provide new approaches to reproductive technologies on Earth. Changes in the speed of mouse spermatozoa after 30 min exposure to simulated weightlessness (by 3D-clinostat) and 2 g hypergravity (by centrifugation) were studied using inhibitory analysis. Simulated microgravity after 30 min led to an increase in the speed of spermatozoa and against the background of an increase in the relative calcium content in the cytoplasm. This effect was prevented by the introduction of 6-(dimethylamino) purine, wortmannin, and calyculin A. Hypergravity led to a decrease in the speed of spermatozoa movement, which was prevented by sodium orthovanadate and calyculin A. At the same time, under microgravity conditions, there was a redistribution of proteins forming microfilament bundles between the membrane and cytoplasmic compartments and under hypergravity conditions—proteins forming networks. The obtained results indicate that even a short exposure of spermatozoa to altered gravity leads to the launch of mechanotransduction pathways in them and a change in motility. Full article

The impact of space radiation and microgravity on DNA damage responses has been discussed controversially, largely due to the variety of model systems engaged. Here, we performed side-by-side analyses of human hematopoietic stem/progenitor cells (HSPC) and peripheral blood lymphocytes (PBL) cultivated in a 2D clinostat to simulate microgravity before, during and after photon and particle irradiation. We demonstrate that simulated microgravity (SMG) accelerates the early phase of non-homologous end joining (NHEJ)-mediated repair of simple, X-ray-induced DNA double-strand breaks (DSBs) in PBL, while repair kinetics in HSPC remained unaltered. Repair acceleration was lost with increasing LET of ion exposures, which increases the complexity of DSBs, precluding NHEJ and requiring end resection for successful repair. Such cell-type specific effect of SMG on DSB repair was dependent on the NF-кB pathway pre-activated in PBL but not HSPC. Already under unperturbed growth conditions HSPC and PBL suffered from SMG-induced replication stress associated with accumulation of single-stranded DNA and DSBs, respectively. We conclude that in PBL, SMG-induced DSBs promote repair of radiation-induced damage in an adaptive-like response. HSPC feature SMG-induced single-stranded DNA and FANCD2 foci, i.e., markers of persistent replication stress and senescence that may contribute to a premature decline of the immune system in space. Full article

Small-cell lung cancer is a fast-growing carcinoma with a poor prognosis and a high level of relapse due to multi-drug resistance (MDR). Genetic mutations that lead to the overexpression of efflux transporter proteins can contribute to MDR. In vitro cancer models play a tremendous role in chemotherapy development and the screening of possible anti-cancer molecules. Low-cost and simple in vitro models are normally used. Traditional two-dimensional (2D) models have numerous shortcomings when considering the physiological resemblance of an in vivo setting. Three-dimensional (3D) models aim to bridge the gap between conventional 2D models and the in vivo setting. Some of the advantages of functional 3D spheroids include better representation of the in vivo physiology and tumor characteristics when compared to traditional 2D cultures. During this study, an NCI-H69AR drug-resistant mini-tumor model (MRP1 hyperexpressive) was developed by making use of a rotating clinostat bioreactor system (ClinoStar^®; CelVivo ApS, Odense, Denmark). Spheroid growth and viability were assessed over a 25-day period to determine the ideal experimental period with mature and metabolically stable constructs. The applicability of this model for anti-cancer research was evaluated through treatment with irinotecan, paclitaxel and cisplatin for 96 h, followed by a 96 h recovery period. Parameters measured included planar surface area measurements, estimated glucose consumption, soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase levels, histology and efflux transporter gene expression. The established functional spheroid model proved viable and stable during the treatment period, with retained relative hyperexpression of the MRP1 efflux transporter gene but increased expression of the P-gp transporter gene compared to the cells cultured in 2D. As expected, treatment with the abovementioned anti-cancer drugs at clinical doses (100 mg/m² irinotecan, 80 mg/m² paclitaxel and 75 mg/m² cisplatin) had minimal impact on the drug-resistant mini-tumors, and the functional spheroid models were able to recover following the removal of treatment. Full article

Background: Simulated microgravity (SMG) has not been well characterized in terms of its impact on cell division structures. This research aimed to assess the changes in cell division in Chang liver cells (CCL-13 cells) under SMG conditions. Methods: CCL-13 cells were exposed to SMG conditions via a 3D clinostat for 72 h. The cells from the control group were kept under the same conditions, without exposure to SMG. The changes in cell division were assessed via cell cycle progression analysis, the transcript expression of the genes associated with the cell cycle, and the appearance of the contractile ring, microvilli, and spindle in CCL-13 cells. Results: The CCL-13 cells from both the control group and the SMG group exhibited a typical epithelial-like shape. The CCL-13 cells of both groups displayed normal nuclear morphologies and were devoid of fragmentation and condensation, which are signs of apoptosis. There were changes in the cell cycle of CCL-13 cells in the SMG condition, which were shown via an increase in the cell percentage in the G0/G1 phase and a decrease in the S phase and G2/M phase. The cell area of the SMG-exposed CCl-13 cells increased, while their nuclear area decreased, which led to a reduction in the nuclear/cytoplasmic ratio. Moreover, the transcript expression of cyclin b1, cyclin d1, cdk2, and cdk6 was downregulated in CCL-13 cells under SMG conditions compared to the control group. Interestingly, SMG-exposed CCL-13 cells exhibited a decreased appearance of microvilli, changes in the formation of the contractile ring, and polar spindle microtubules during cytokinesis. Conclusions: SMG attenuated the cell division of CCL-13 cells by driving cells into the arrest phase and altering the cell division structures. Full article

Weightlessness in space leads to bone loss, muscle atrophy, and impaired immune defense in astronauts. Mesenchymal stem cells (MSCs) play crucial roles in maintaining the homeostasis and function of the tissue. However, how microgravity affects the characteristics MSCs and the related roles in the pathophysiological changes in astronauts remain barely known. Here we used a 2D-clinostat device to simulate microgravity. Senescence-associated-β-galactosidase (SA-β-gal) staining and the expression of senescent markers p16, p21, and p53 were used to evaluate the senescence of MSCs. Mitochondrial membrane potential (mΔΨm), reactive oxygen species (ROS) production, and ATP production were used to evaluate mitochondrial function. Western blot and immunofluorescence staining were used to investigate the expression and localization of Yes-associated protein (YAP). We found that simulated microgravity (SMG) induced MSC senescence and mitochondrial dysfunction. Mito-TEMPO (MT), a mitochondrial antioxidant, restored mitochondrial function and reversed MSC senescence induced by SMG, suggesting that mitochondrial dysfunction mediates SMG-induced MSC senescence. Further, it was found that SMG promoted YAP expression and its nuclear translocation in MSCs. Verteporfin (VP), an inhibitor of YAP, restored SMG-induced mitochondrial dysfunction and senescence in MSCs by inhibiting YAP expression and nuclear localization. These findings suggest that YAP inhibition alleviates SMG-induced MSC senescence via targeting mitochondrial dysfunction, and YAP may be a potential therapeutic target for the treatment of weightlessness-related cell senescence and aging. Full article

A diabetogenic state induced by spaceflight provokes stress and health problems in astronauts. Microgravity (µg) is one of the main stressors in space causing hyperglycaemia. However, the underlying molecular pathways and synergistic effects of µg and hyperglycaemia are not fully understood. In this study, we investigated the effects of high glucose on EA.hy926 endothelial cells in simulated µg (s-µg) using a 3D clinostat and static normogravity (1g) conditions. After 14 days of cell culture under s-µg and 1g conditions, we compared the expression of extracellular matrix (ECM), inflammation, glucose metabolism, and apoptosis-related genes and proteins through qPCR, immunofluorescence, and Western blot analyses, respectively. Apoptosis was evaluated via TUNEL staining. Gene interactions were examined via STRING analysis. Our results show that glucose concentrations had a weaker effect than altered gravity. µg downregulated the ECM gene and protein expression and had a stronger influence on glucose metabolism than hyperglycaemia. Moreover, hyperglycaemia caused more pronounced changes in 3D cultures than in 2D cultures, including bigger and a greater number of spheroids, upregulation of NOX4 and the apoptotic proteins NF-κB and CASP3, and downregulation of fibronectin and transglutaminase-2. Our findings bring new insights into the possible molecular pathways involved in the diabetogenic vascular effects in µg. Full article

Microgravity is a novel strategy that may serve as a complementary tool to develop future cancer therapies. In lung cancer, the influence of microgravity on cellular processes and the migratory capacity of cells is well addressed. However, its effect on the mechanisms that drive lung cancer progression remains in their infancy. In this study, 13 differentially expressed genes were shown to be associated with the prognosis of lung cancer under simulated microgravity (SMG). Using gene set enrichment analysis, these genes are enriched in humoral immunity pathways. In lieu, alveolar basal-epithelial (A549) cells were exposed to SMG via a 2D clinostat system in vitro. In addition to morphology change and decrease in proliferation rate, SMG reverted the epithelial-to-mesenchymal transition (EMT) phenotype of A549, a key mechanism in cancer progression. This was evidenced by increased epithelial E-cadherin expression and decreased mesenchymal N-cadherin expression, hence exhibiting a less metastatic state. Interestingly, we observed increased expression of FCGBP, BPIFB, F5, CST1, and CFB and their correlation to EMT under SMG, rendering them potential tumor suppressor biomarkers. Together, these findings reveal new opportunities to establish novel therapeutic strategies for lung cancer treatment. Full article

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. Full article

Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into dermal fibroblasts to participate in skin-repairing. However, at present, little is known about how microgravity affects dermal fibroblastic differentiation of BMSCs in space. The aim of this study was to investigate the effect of simulated microgravity (SMG) on the differentiation of BMSCs into dermal fibroblasts and the related molecular mechanism. Here, using a 2D-clinostat device to simulate microgravity, we found that SMG inhibited the differentiation and suppressed the Wnt/β-catenin signaling and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2). After upregulating the Wnt/β-catenin signaling with lithium chloride (LiCl) treatment, we found that the effect of the differentiation was restored. Moreover, the Wnt/β-catenin signaling was upregulated when phosphorylation of ERK1/2 was activated with tert-Butylhydroquinone (tBHQ) treatment. Taken together, our findings suggest that SMG inhibits dermal fibroblastic differentiation of BMSCs by suppressing ERK/β-catenin signaling pathway, inferring that ERK/β-catenin signaling pathway may act as a potential intervention target for repairing skin injury under microgravity conditions. Full article

Simulated microgravity (SMG) induced the changes in cell proliferation and cytoskeleton organization, which plays an important factor in various cellular processes. The inhibition in cell cycle progression has been considered to be one of the main causes of proliferation inhibition in cells under SMG, but their mechanisms are still not fully understood. This study aimed to evaluate the effects of SMG on the proliferative ability and cytoskeleton changes of Chang Liver Cells (CCL-13). CCL-13 cells were induced SMG by 3D clinostat for 72 h, while the control group were treated in normal gravity at the same time. The results showed that SMG reduced CCL-13 cell proliferation by an increase in the number of CCL-13 cells in G0/G1 phase. This cell cycle phase arrest of CCL-13 cells was due to a downregulation of cell cycle-related proteins, such as cyclin A1 and A2, cyclin D1, and cyclin-dependent kinase 6 (Cdk6). SMG-exposed CCL-13 cells also exhibited a downregulation of α-tubulin 3 and β-actin which induced the cytoskeleton reorganization. These results suggested that the inhibited proliferation of SMG-exposed CCL-13 cells could be associate with the attenuation of major cell cycle regulators and main cytoskeletal proteins. Full article

Clinorotation was the first method designed to simulate microgravity on ground and it remains the most common and accessible simulation procedure. However, different experimental settings, namely angular velocity, sample orientation, and distance to the rotation center produce different responses in seedlings. Here, we compare A. thaliana root responses to the two most commonly used velocities, as examples of slow and fast clinorotation, and to vertical and horizontal clinorotation. We investigate their impact on the three stages of gravitropism: statolith sedimentation, asymmetrical auxin distribution, and differential elongation. We also investigate the statocyte ultrastructure by electron microscopy. Horizontal slow clinorotation induces changes in the statocyte ultrastructure related to a stress response and internalization of the PIN-FORMED 2 (PIN2) auxin transporter in the lower endodermis, probably due to enhanced mechano-stimulation. Additionally, fast clinorotation, as predicted, is only suitable within a very limited radius from the clinorotation center and triggers directional root growth according to the direction of the centrifugal force. Our study provides a full morphological picture of the stages of graviresponse in the root tip, and it is a valuable contribution to the field of microgravity simulation by clarifying the limitations of 2D-clinostats and proposing a proper use. Full article

Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer. Full article