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.

Alterations in the activity and sleep of Drosophila melanogaster under simulated microgravity

This study aimed to investigate alterations in the activity and sleep of Drosophila melanogaster under simulated microgravity, which was implemented through the random positioning machine, while different light conditions (normal photoperiod and constant dark) were set. Fruit flies of different strains and sexes were treated for 3 days, and activity and sleep were monitored using the Drosophila Activity Monitoring System. After 3 days of treatment, fruit flies were sampled to detect the relative expression levels of the major clock genes and some neurotransmitter-related genes. The results showed that for the normal photoperiod (LD) condition, the activity increased and sleep decreased under simulated microgravity, while for the constant dark (DD) condition, the activity and sleep rhythms appeared disordered and the activity increased, thus decreasing the likelihood of waking up during the day. Light conditions, strains, and sexes, individually or in combination, had impacts on the simulated microgravity effects on behaviors. The clock genes and neurotransmitter-related genes had different degrees of response among sexes and strains, although the overall changes were slight. The results indicated that the normal photoperiod could ease the effects of simulated microgravity on fruit flies’ activity and sleep and possible unidentified pathways involved in the regulatory mechanism need further exploration. This study is expected to provide ideas and references for studying the effects of microgravity on space life science.

Mouse and Fly Sperm Motility Changes Differently under Modelling Microgravity

Sperm motility is essential for the natural fertilization process in most animal species. Despite the fact that evolution took place under conditions of constant gravity, the motility of spermatozoa of insects and mammals under microgravity conditions changes in different ways. In this work, an attempt was made to explain this effect. The sperm motility of the fruit fly Drosophila melanogaster and the mouse was evaluated after exposure to a random positioning machine for 6 h. Sodium fluoride was used to inhibit serine/threonine phosphatases, sodium orthovanadate was used to inhibit tyrosine phosphatases, and 6-(dimethylamino)purine was used to inhibit protein kinases. The results obtained indicate that simulated microgravity leads to an increase in the speed of movement of fly spermatozoa by 30% (p < 0.05), and this effect is blocked by sodium fluoride. In contrast, a 29% (p < 0.05) decrease in the speed of movement of mouse spermatozoa under simulated microgravity is prevented by 6-(dimethylamino)purine. Moreover, after 6 h of exposure, the content of tubulin cytoskeleton and actin proteins remains at the control level in the spermatozoa of flies and mice. However, the content of the actin-binding protein alpha-actinin in fly sperm decreases by 29% (p < 0.05), while in mouse sperm, the relative content of alpha-actinin1 increases by 94% (p < 0.05) and alpha-actinin4 by 121% (p < 0.05) relative to the control, as determined by 6 simulated microgravity tests. It can be assumed that the effect of simulated microgravity on the motility of mammalian spermatozoa is mediated through the regulation of phosphorylation and that of insects through the regulation of dephosphorylation of motor proteins; moreover, the development of a response to changes in external mechanical conditions has a different time scale.

Alterations of Growth and Focal Adhesion Molecules in Human Breast Cancer Cells Exposed to the Random Positioning Machine

In this study, we evaluated changes in focal adhesions (FAs) in two types of breast cancer cell (BCC) lines (differentiated MCF-7 and the triple-negative MDA-MB-231 cell line) exposed to simulated microgravity (s-μg) created by a random positioning machine (RPM) for 24 h. After exposure, the BCC changed their growth behavior and exhibited two phenotypes in RPM samples: one portion of the cells grew as a normal two-dimensional monolayer [adherent (AD) BCC], while the other portion formed three-dimensional (3D) multicellular spheroids (MCS). After 1 h and 30 min (MDA-MB-231) and 1 h 40 min (MCF-7), the MCS adhered completely to the slide flask bottom. After 2 h, MDA-MB-231 MCS cells started to migrate, and after 6 h, a large number of the cells had left the MCS and continued to grow in a scattered pattern, whereas MCF-7 cells were growing as a confluent monolayer after 6 h and 24 h. We investigated the genes associated with the cytoskeleton, the extracellular matrix and FAs. ACTB, TUBB, FN1, FAK1, and PXN gene expression patterns were not significantly changed in MDA-MB-231 cells, but we observed a down-regulation of LAMA3, ITGB1 mRNAs in AD cells and of ITGB1, TLN1 and VCL mRNAs in MDA-MB-231 MCS. RPM-exposed MCF-7 cells revealed a down-regulation in the gene expression of FAK1, PXN, TLN1, VCL and CDH1 in AD cells and PXN, TLN and CDH1 in MCS. An interaction analysis of the examined genes involved in 3D growth and adhesion indicated a central role of fibronectin, vinculin, and E-cadherin. Live cell imaging of eGFP-vinculin in MCF-7 cells confirmed these findings. β-catenin-transfected MCF-7 cells revealed a nuclear expression in 1g and RPM-AD cells. The target genes BCL9, MYC and JUN of the Wnt/β-catenin signaling pathway were differentially expressed in RPM-exposed MCF-7 cells. These findings suggest that vinculin and β-catenin are key mediators of BCC to form MCS during 24 h of RPM-exposure.

Molecular Genetic Analysis of Neural Stem Cells After Space Flight And Simulated Microgravity on Earth

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (bNCSCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of co-cultured and co-implanted cells. bNCSCs were launched to space (Space cells), onboard Sounding rocket as free-floating neurospheres or in bioprinted scaffold. For comparison, bNCSCs were placed in random positioning machine to simulate microgravity (Microgravity cells) or cultured under Earth conditions. Using Next-Generation RNA sequencing and data post-processing, a list of genes that were at least two-fold changed between control cells and Space cells were selected for further analysis. Functional clusters of enriched genes were obtained by gene ontology bioinformatics, using the DAVID program, and Ingenuity Pathway Analysis was used to predict functional implications of the identified gene expressions. Space cells upregulated genes related to proliferation and survival, whereas Microgravity cells upregulated genes associated with differentiation and inflammation. Thus, i) space flight provides unique conditions with distinctly different effects on the properties of bNCSCs compared to Earth controls, and ii) may induce post-flight properties that reinforce the utility of bNCSCs for regenerative medicine and tissue engineering.

Tissue Engineering of Cartilage Using a Random Positioning Machine

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.