Observation of host cell morphological changes under simulated-microgravity conditions

Observation of host cell morphological changes under simulated-microgravity conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Observation of host cell morphological changes under simulated-microgravity conditions Takafumi Yamada, Hiroki Bochimoto, Daisuke Kondoh, Fumi Murakoshi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6810961/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Microgravity exposure has been associated with various health concerns, including reports of opportunistic infections. However, the specific changes that occur in host cells under these conditions remain unclear. In this study, we investigated the effect of microgravity on Vero cells and human foreskin fibroblasts (HFFs), which serve as host cells. Under simulated-microgravity conditions, both Vero cells and HFFs formed multicellular spheroids, representing an irreversible change to the viable cells. Moreover, Vero cell proliferation after 7 days of culture under simulated-microgravity conditions was increased compared to normal gravity conditions. Electron microscopy revealed widened intercellular spaces under the simulated-microgravity conditions, indicating morphological changes. RNA sequencing showed no substantial changes in the expression levels of genes related to growth ability. However, some changes in gene expression support the phenomena like reductions in collagen levels, which are reported previously. Our findings suggest that simulated microgravity influences cell morphology and proliferation, and offer insights into maintaining the health of astronauts. Biological sciences/Cell biology/Cell growth Health sciences/Risk factors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction As space development progresses, more and more people are expected to stay in space in the future. However, humans in space experience greater stressors compared to those on Earth, primarily due to factors like microgravity, radiation exposure, and confinement within a closed environment. These stressors contribute to muscle and bone loss 1 , 2 , tissue inflammation 3 , central nervous system disorders 4 , and various other health risks. Furthermore, some astronauts have developed opportunistic infections with viruses such as cytomegalovirus 5 or Herpes virus 6 during their stay on the International Space Station (ISS) due to decreased immune function. In fact, there have been 29 reported infections among a total of 742 crew members in the space shuttle program so far 7 . While detailed studies of infection in microgravity are necessary to explore various opportunistic pathogens, there is limited knowledge about how these pathogens behave within the host cells. To date, animal cell culture microgravity-simulation studies have been conducted on only a limited number of cell types such as stem cells 8 , skin cells 9 , and cancer cells 10 . An in-depth understanding of the effects of microgravity on host cells is not only needed for future research on infectious diseases, but also to help protect the health of astronauts. This study focuses on two types of host cells, Vero cells and human foreskin fibroblasts (HFFs), which can serve as models of host response during infection with opportunistic pathogens and observe their behavior under conditions that simulate a microgravity environment. Vero cells are cultured cells derived from the kidneys of an African green monkey and have been widely employed in global microbiology research, pathogen testing in medical settings, and viral vaccine production 11 . Vero cells lack interferon production and are susceptible to a wide range of viruses; however, due to this limitation, they cannot adequately represent human cells in terms of immune response to pathogens. Therefore, it is essential to compare findings from Vero cells with those from primary cell lines such as HFFs. Herpes virus, a concern for astronaut infections, has been cultured in both Vero cells 12 and HFFs 13 . Therefore, these cell types were selected to study viral infections in microgravity. We create a simulated microgravitational environment by using a 3D clinostat (Advanced Engineering Services co., ltd.). This device rotates a flask filled with medium, and the gravity on cells is distributed. It has been utilized in multiple studies, and its effectiveness has been demonstrated 14 , 15 . For example, skin cells cultured in a 3D clinostat exhibit a reduction in collagen, like the skin cells of astronauts after a space mission 9 . Compared to the microgravity experiments conducted on the ISS or in aircraft, it is possible to create a stable environment inexpensively with a 3D clinostat. Our study examined the effects of simulated microgravity by analyzing the number, morphology, and variation in expression genes of host cells. Our findings will lead to further investigation into the effects of microgravity on host cells, to analyze the causes of opportunistic infections. Materials and methods Cells Vero cells (provided by Dr. Y. Kawaguchi) were maintained in Dulbecco Minimal Essential Medium (DMEM: Nacalai Tesque) supplemented with 5% or 2% fetal bovine serum (FBS) and penicillin (100 U/ml) -streptomycin (100 µg/ml) solution (Nacalai Tesque). Human foreskin fibroblasts (HFFs) (ATCC: SCRC-1041) were maintained in RPMI 1640 medium (Nacalai Tesque) supplemented with 5% or 2% fetal bovine serum (FBS) and penicillin (100 U/ml) -streptomycin (100 µg/ml) solution (Nacalai Tesque). The cells are cultured in 5% FBS until the flask reaches 80% confluent. They are maintained in a 2% FBS environment under simulated microgravity environment. All cultures were maintained at 37°C under 5% CO 2 . Simulated microgravity Cells were subjected to a microgravitational environment by use of a 3D clinostat (Advanced Engineering Services co., ltd.) (Supplementary Fig. 1a). They rotated in an incubator kept at 37°C under 5% CO 2 . The average gravitational force acting on the cells was reduced to about 10^-3 when the clinostat rotated at 30 rpm 8 . We used iP-TEC® Flasks (25-cm 2 ) with a vent cap (Supplementary Fig. 1b) to rotate the samples in the clinostat. These flasks have a semi-permeable cap and were fully filled with medium during the experiments. The flasks were fixed to the clinostat with a band. Several flasks could be kept in the same incubator so that comparative experiments could be also performed. In this research, every sample was first cultured in normal gravity for 2 days. Then, the cells were transferred to the 3D clinostat to start the culture (day 0); at this point, they were 80% confluent. The cells were cultured in simulated microgravity for 7 days. Electron Microscopy Vero cells (cultured under normal gravity and simulated microgravity for 7 days) were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4, 4°C, then gently removed from the flasks by using a cell scraper and pelleted via centrifugation (1000rpm, 5min). The pellets were thoroughly rinsed with 0.1 M PB. Then the pellets were fixed with 1% OsO 4 in 0.1 M PB (pH 7.4) for 2 h at 4°C. The fixed pellets were then rinsed with 70% ethanol, dehydrated through a graded ethanol series and propylene oxide, and embedded in Epon 812. Ultrathin sections (80-nm-thick) were cut using an EM UC7i ultramicrotome (Leica microsystems) and examined using an HT7700 transmission electron microscope (Hitachi) without uranyl acetate or lead citrate staining. The brightness and contrast of the obtained TIFF data were adjusted by using Photoshop CS6 (Adobe). RNA sequencing analysis Vero cell (cultured under normal gravity and simulated microgravity for 7 days) mRNA was purified by using a FastGene™ RNA Premium Kit (50 preps: Nippon Genetics) for RNA sequencing (Transcriptome). RNA sequencing was performed by BGI Japan. Paired-end libraries were sequenced using the DNBSEQ- (2 × 150 nucleotide read length). platform, which generated about 6.73G Gb bases per sample. The average mapping ratio with the reference genome was 97.68%, and the average mapping ratio with the reference gene was 77.32%. Bioinformatics analyses were performed using BGI's proprietary Dr. TOM software and BGI's in-house customized data mining system ( https://biosys.bgi.com ). Staining Sample cells were stained with 50 µg/ml propidium iodide (PI: Nacalai Tesque) solution (Absorbance: 40-fold dilution, water λ max: around 492 nm). Dead cells fluoresced red when incubated at room temperature for 5 minutes in the dark. Statistical Analyses Student's t-test was performed for comparisons between two normally distributed samples, using the statistical analysis function in Microsoft Excel. Differences between the experimental groups were considered significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. Data are expressed as the mean ± SE of the mean (SEM). Results In a simulated-microgravity environment, Vero cells form three-dimensional aggregates. We cultured Vero cells in normal gravity and in the simulated microgravity for 7 days. In the microgravity environment, Vero cells formed several three-dimensional aggregates known as multicellular spheroids (MCSs) 15 . In contrast, Vero cells cultured under normal gravity maintained a sheet-like morphology (Fig. 1 a). Higher magnification images of these cells on day 7 further detailed their respective morphologies under both conditions (Fig. 1 b). To examine changes immediately after transfer to microgravity, we observed Vero cells every 2 h after they were placed in the simulated-microgravity environment. We observed that the MCSs formed rapidly, within 4 to 6 h (Fig. 1 c). Magnified views of the cells at the 4h and 6h time points illustrate this aggregation process (Fig. 1 d). We then compared Vero cells cultured under simulated microgravity for 24 h with those cultured under simulated microgravity for 24 h followed by normal gravity conditions for another 24 h. The results revealed that the formation of the MCSs was irreversible (Fig. 1 e). Of note, these MCSs primarily consisted of live cells, as evidenced by partial staining with the PI solution (Fig. 1 f). While our analysis primarily focused on Vero cells as a representative cell line, it is imperative to investigate the effects of simulated microgravity on primary cells to ascertain whether similar phenotypic changes occur. Consequently, we conducted analogous experiments with human foreskin fibroblasts (HFFs). After 7 days of culture, HFFs exhibited structures resembling MCSs, although smaller than those observed with Vero cells (Fig. 2 ). Unlike the Vero cell MCSs, which appeared to consist of tens of cells, the HFF MCSs comprised only 2–3 cells based on optical microscopy observations. Further analysis using transmission electron microscopy (TEM) revealed changes in cell thickness and intercellular spacing for both Vero cells (Fig. 3 a) and HFFs (Fig. 3 b). Simulated microgravity promotes cell proliferation. The number of cells after 7 days of culture was measured for flasks that initially contained the same number of cells. A significant increase in the number of Vero cells under simulated microgravity, with an approximately 1.6-fold difference in cell numbers, was observed (Fig. 4 a). For HFFs, the number of cells was compared between static culture and microgravity after 7 days of culture. HFFs also showed an approximately 1.2-fold increase in cell numbers, although statistical significance was not confirmed by t-test results (Fig. 4 b). In addition to static culture and simulated microgravity, to investigate the effects of cell directions and gravity, we prepared flasks in upside down and sideways condition (Fig. 4 c). Sheet-like cultured cells were cultivated for 7 days under four conditions: normal gravity standing, normal gravity sideways, normal gravity upside-down, and simulated microgravity. MCS formation and increased cell numbers were observed only under simulated microgravity. Changes in gene expression levels. To determine how microgravity affects gene expression, a comparative analysis of mRNA expression in cells cultured under normal gravity and microgravity was performed. Gene Ontology (GO) Classification (Fig. 5 a), KEGG pathway classification (Fig. 5 b), and a Volcano plot (Fig. 5 c) of the results are shown. In cells cultured under microgravity, 18 Differentially Expressed Genes (DEGs) were upregulated and 106 DEGs were downregulated (Q-value 1) compared to cells cultured under normal gravity (Supplementary table 1 ). In the GO classification, the X-axis represents the number of genes annotated to GO terms, and the Y-axis represents the category of GO terms. Each bar shows the number of genes with similar functions. In the KEGG pathway classification, performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 16 , 17 , the vertical axis is the number of genes annotated to a category of the KEGG Pathway, and the horizontal axis is the category of KEGG Pathway. In the volcano plot, the vertical axis represents the statistical significance of the data, whereas the horizontal axis indicates the expression ratio. Red represents upregulated DEGs, green represents downregulated DEGs, and gray represents non-DEGs. From the result of GO classification, we identified 14 DEGs involved in cell proliferation. We observed two patterns: an increase in cell proliferation-promoting factors or a decrease in cell proliferation-inhibiting factors, and conversely, a decrease in cell proliferation-promoting factors or an increase in cell proliferation-inhibiting factors. Of the 14 DEGS, the five that showed the most significant changes in expression level (i.e., ADM, HILPDA, STC1, BAMBI , and IL6 ) were decreases in cell proliferation-promoting factors 18 , 19 , 20 , 21 , 22 . We also obtained 15 DEGs involved in the immune system. Of these genes, the five most noteworthy, which all decreased, were LOX, CCN4, IL6 , and BNIP3 ), which are involved in the formation and survival of malignant tumors, and PTX3 , which is involved in humoral innate immunity 23 , 24 , 25 , 26 , 27 . Discussion In this study, we found that simulated microgravity has an impact on the morphology and proliferation of cells. Vero cells showed proliferation with statistical significance, and for the HFFs, the t-test yielded a P -value of approximately 0.13, suggesting potential variability in the values. Previous research has suggested that alterations in the cytoskeleton and extracellular matrix may cause MCS formation 15 . Our electron microscopy findings support this hypothesis. As the MCSs increase over time, it suggests that MCSs are formed through the stacking of divided cells in a spherical configuration. However, the increased Vero cell numbers cannot be solely attributed to MCS formation due to their sparse density. Insights into the increase in cell numbers can be gleaned from the electron microscopy images (Fig. 3 ). Cells observed under the electron microscope appeared detached and suspended from the flask surface. Therefore, it is likely that the images depict cells in a sheet-like rather than MCS formation, as indicated by the high density of stacked cells, allowing more cells to be accommodated to restricted space. Similar reasoning applies to the HFF electron microscopy images, supporting the notion that simulated microgravity induces three-dimensional structural changes in sheet of cells. Based on our results from the four conditions tested (i.e., normal gravity standing, normal gravity sideways, normal gravity upside-down, and simulated microgravity), we infer that simulated microgravity induces an effect that does not merely reflect a change the direction of gravity acting on cells. The RNA sequencing results from Vero cells revealed differences in gene expression between static cultivation and simulated-microgravity conditions. The GO classification (Fig. 5 a) indicated a decrease in cell proliferation factors, and we were unable to obtain evidence that cell proliferation was genetically promoted. In contrast, the KEGG pathway Classification (Fig. 5 b) revealed a significant decrease in the expression of TNFRSF10B , which is part of the apoptosis-inducing pathway 28 within the “cell growth and death” category. This could potentially inhibit apoptosis, thereby leading to an increase in cell numbers. From the GO classification, the expression of two prominent genes ( CXCR4 , and NDRG1 ) associated with cell junctions was found to be downregulated. The upregulation in CLDN2 may be an adaptive response to weakened cell adhesion. Although not included among the genes associated with cell junctions, we also observed an decrease in the expression of growth differentiation factors (e.g., GDF11 ) that are involved in collagen secretion. Our results suggest a potential weakening of cell adhesion and adaptive responses, consistent with previously reported reductions in collagen levels 9 . The statistically significant decrease in the expression of the exocytosis-enhancing factor ( NDRG1 ) also indicates a possible link to the decline in immune function under microgravity conditions 29 , 30 . Additionally, we examined other genes that showed notable differences in expression levels in a volcano plot (Fig. 5 c). The most upregulated gene, TMEM151A , plays a role in paroxysmal movement disorders 31 , but its relevance here is unclear. NAT8 and ISM1 , which were the second and third most upregulated genes, respectively, have been reported to assist in viral replication 32 and to modulate antiviral activity 33 , suggesting a potential involvement in immune responses. In summary, in this study, we detected changes in host cells under simulated-microgravity conditions through morphological observations, cell count measurements, and RNA sequencing. Our findings from electron microscopy suggest that these cellular changes may be influenced by not only endocrine factors but also physical forces. The implications of our findings for pathogen growth remain unclear. Nonetheless, it is essential to consider such changes in host cells when investigating infectious diseases under microgravity conditions. The findings from this study provide a novel perspective on opportunistic infections during space missions. Declarations Competing interests All authors declare no financial competing interests. Funding Declaration This research was supported by a Grant-in-Aid for Scientific Research (B: 24K01921) from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan, by a Livestock Promotional Subsidy from the Japan Racing Association, and by Front Loading Research from the Advisory Committee for Space Utilization Research in the Japan Aerospace Exploration Agency (JAXA) and the Institute of Space and Astronautical Science (ISAS). Author Contribution T.Y. designed the study, collected data and drafted the manuscript. H.B. and D.K. contributed to collecting data and helped draft the manuscript. F.M. contributed to data analysis and edited the manuscript. K.K. supervised the project and edited the manuscript. Data Availability The datasets generated and analysed during the current study are available in the Sequence Read Archive (SRA) repository, under the BioProject accession number PRJNA1283931 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1283931). References Nagaraja, M. P. & Risin, D. The current state of bone loss research: data from spaceflight and microgravity simulators. J. Cell. Biochem. 114 , 1001–1008 (2013). Blaber, E., Marçal, H. & Burns, B. P. Bioastronautics: the influence of microgravity on astronaut health. Astrobiology 10 , 463–473 (2010). Patel, Z. S. et al. Red risks for a journey to the red planet: The highest priority human health risks for a mission to Mars. npj Microgravity . 6 (1), 33 (2020). Kokhan, V. S., Shakhbazian, E. V. & Markova, N. A. Psycho-emotional status but not cognition is changed under the combined effect of ionizing radiations at doses related to deep space missions. Behav. Brain. Res. 362 , 311–318 (2019). Mehta, S. K. et al. Reactivation and shedding of cytomegalovirus in astronauts during spaceflight. The J. Infect. diseases . 182 , 6 1761–1764 (2000). Rooney, B. V. et al. Herpes virus reactivation in astronauts during spaceflight and its application on earth. Front. Microbiol. 10 , 432964 (2019). Crucian, B., Kunz, H. & Clarence, F. Sams. Risk of crew adverse health event due to altered immune response. No. JSC-CN-33468. (2015). Pisanu, M. et al. Lung cancer stem cell lose their stemness default state after exposure to microgravity. BioMed Res. international 2014 (2014). Choi, D. et al. 3D cell culture using a clinostat reproduces microgravity-induced skin changes. npj Microgravity . 7 (1), 20 (2021). Ahn, J. et al. Morphologic and genomic changes of thyroid cancer cell lines exposed to conditions of simulated microgravity. npj Microgravity . 10 (1), 8 (2024). Sheets, R. History and characterization of the Vero cell line. Cent Biol. Eval Res 1–12 (2000). Shen, C. F. et al. Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus‐1. Biotechnol. J. 19.1 , 2300244 (2024). Rasmussen, L. U. C. Y. Farley. Inhibition of Herpesvirus hominis replication by human interferon. Infect. Immun. 12.1 , 104–108 (1975). Kim, Y. et al. Time-averaged simulated microgravity (taSMG) inhibits proliferation of lymphoma cells, L-540 and HDLM-2, using a 3D clinostat. Biomed. Eng. Online . 16 , 1–12 (2017). Choi, D. et al. 3D cell culture using a clinostat reproduces microgravity-induced skin changes. npj Microgravity 7.1 20 (2021). Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28 , 27–30 (2000). Kanehisa, M., Furumichi, M., Sato, Y., Kawashima, M. & Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 51 , D587–D592 (2023). Chen et al. Adrenomedullin promotes human endothelial cell proliferation via HIF-1α. Molecular and cellular biochemistry 365 : 263–273. (2012). Sheng, Y. et al. Hypoxia-inducible lipid droplet-associated (HILPDA) facilitates the malignant phenotype of lung adenocarcinoma cells in vitro through modulating cell cycle pathways. Tissue Cell. 70 , 101495 (2021). Bai, Y. et al. Stanniocalcin 1 promotes cell proliferation via cyclin E1/cyclin–dependent kinase 2 in human prostate carcinoma. Oncol. Rep. 37 (4), 2465–2471 (2017). Zhou, L. et al. The overexpression of BAMBI and its involvement in the growth and invasion of human osteosarcoma cells. Oncol. Rep. 30 (3), 1315–1322 (2013). Ogata, A. et al. IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. Journal of immunology (Baltimore, Md.: 159.5 (1997): 2212–2221. (1950). Tenti, P. & Vannucci, L. Lysyl oxidases: linking structures and immunity in the tumor microenvironment. Cancer Immunol. Immunother. 69 (2), 223–235 (2020). Singh, K. & Sunday, S. Oladipupo. An overview of CCN4 (WISP1) role in human diseases. J. Translational Med. 22 (1), 1–24 (2024). Neurath, M. F. & Susetta Finotto IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer. Cytokine Growth Factor Rev. 22.2 , 83–89 (2011). Ney, P. A. Mitochondrial autophagy: Origins, significance, and role of BNIP3 and NIX. Biochim. et Biophys. Acta (BBA)-Molecular Cell. Res. 1853 (10), 2775–2783 (2015). Deban, L. et al. Pentraxins in innate immunity: lessons from PTX3. Cell Tissue Res. 343 (1), 237–249 (2011). Yang, A., Wilson, N. S. & Ashkenazi, A. Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation. Curr. Opin. Cell Biol. 22 (6), 837–844 (2010). Hodgson, R. et al. NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance. Commun. Biology . 5.1 , 1216 (2022). Anna, M. et al. Systems approach reveals distinct and shared signaling networks of the four PGE2 receptors in T cells. Sci. Signal. 14 , eabc8579 (2021). Li, H. F. et al. TMEM151A variants cause paroxysmal kinesigenic dyskinesia. Cell. Discovery . 7 (1), 83 (2021). Zhao, X. et al. N-Acetyltransferase 8 promotes viral replication by increasing the stability of enterovirus 71 nonstructural proteins . J. Virol. 96 , e00119–e00122 (2022). Li, C. et al. Zebrafish Ism1 is a novel antiviral factor that positively regulates antiviral immune responses. Dev. Comp. Immunol. 125 , 104210 (2021). Additional Declarations No competing interests reported. Supplementary Files Supplementarytable.xlsx supplementaryfigure1a.mp4 Slide6.jpg Supplementary Figure 1. Microgravity simulator a. Movie of rotating clinostat (Advanced Engineering Services co., Ltd.). b. iP-TEC® Flasks with a vented cap. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6810961","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":535073583,"identity":"91c415bf-7bc8-4129-89ee-f27f277d05d5","order_by":0,"name":"Takafumi Yamada","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Takafumi","middleName":"","lastName":"Yamada","suffix":""},{"id":535073584,"identity":"ffd0e7e6-5a8f-404c-8937-b567f52cfe06","order_by":1,"name":"Hiroki Bochimoto","email":"","orcid":"","institution":"The Jikei University School of 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11:17:33","extension":"pptx","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40910712,"visible":true,"origin":"","legend":"","description":"","filename":"Figuresscientificreports.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/c5354ba961ee1a831af1ed5b.pptx"},{"id":94756519,"identity":"1974654e-76c2-4766-be8b-c303d2ccd94f","added_by":"auto","created_at":"2025-10-30 11:17:33","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70283,"visible":true,"origin":"","legend":"","description":"","filename":"5dbd1891969c41f698204fa59550bd031structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/9faeb906886b60c34e7b27d0.xml"},{"id":94756518,"identity":"0a12495c-e459-4014-8d0b-46f6630b131b","added_by":"auto","created_at":"2025-10-30 11:17:32","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80407,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/90aade45e8d832ad7ac73b2e.html"},{"id":94823796,"identity":"c3fe1b4a-5f91-4453-8b75-3cae71ed8f69","added_by":"auto","created_at":"2025-10-31 06:47:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMulticellular spheroids (MCSs) form in Vero cells under simulated-microgravity conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Sheet of Vero cells before the experiment, and Vero cells cultured in normal gravity and simulated microgravity for 7 days. The arrows indicate the MCSs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Magnified views of Vero cells cultured in normal gravity and simulated microgravity for 7 days. The arrows indicate the MCSs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003e Sheet of Vero cells before the experiment, and Vero cells cultured in simulated microgravity for 2, 4, and 6 h continuously. The arrows indicate the MCSs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e Magnified views of Vero cells cultured in simulated microgravity for 4 and 6 h, The arrows indicate the MCSs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003e Vero cells cultured under simulated microgravity for 24 h, and Vero cells cultured under normal gravity for 24 h after being cultured under simulated microgravity for 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef.\u003c/strong\u003e Vero cells cultured under normal gravity, and simulated microgravity for 24 h; Stained with propidium iodide.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/9ece3dff504f99463545955c.jpg"},{"id":94756506,"identity":"21c5ef82-386b-4117-b00b-24db19df565c","added_by":"auto","created_at":"2025-10-30 11:17:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMCSs form HFFs under simulated-microgravity conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSheet of HFFs before the experiment, and HFFs cultured in normal gravity and simulated microgravity for 7 days. The arrows indicate the MCSs.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/7de6fe73966618950aeccaad.jpg"},{"id":94756512,"identity":"53f2c371-50c4-4480-bb86-487df8be17a0","added_by":"auto","created_at":"2025-10-30 11:17:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrastructural changes and stratification of cultured Vero cells (A) and HFFs (B) under simulated microgravity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA layer of cultured cells consists of 2–3 thin cells under normal gravity, but 5–6 thick cells under microgravity. Cells adhered tightly in continuous planes under normal gravity, but loosely in some spots under microgravity. Arrowheads indicate cell adhesion sites. Boxes in the upper panels correspond to the areas shown in the lower panels.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/fad68d05f8a67783104326bd.jpg"},{"id":94823168,"identity":"4452e63f-850d-4116-ad4a-e266d21b115e","added_by":"auto","created_at":"2025-10-31 06:46:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the number of cells after 7 days of culture.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eNumber of Vero cells after culturing for 7 days under normal gravity or simulated microgravity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eNumber of HFFs after culturing for 7 days under normal gravity or simulated microgravity.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; The cells were counted using a hemocytometer. The unit of data is per flask (25 cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ratio of Vero cell counts after 7 days of culture under normal gravity (i.e., static, sideways, and upside-down) and under simulated microgravity.\u003c/p\u003e\n\u003cp\u003eThe cells were counted using a hemocytometer.\u003c/p\u003e\n\u003cp\u003eBrackets over the point columns represent significant differences by the Student's t-test: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/5c5a1013f93940be921e93d7.jpg"},{"id":94756514,"identity":"9436dd05-4bd6-4a61-938d-5180272d73a0","added_by":"auto","created_at":"2025-10-30 11:17:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes in Vero cells under normal gravity and simulated microgravity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e GO pathway classification of Vero cells indicating the number of gene types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e KEGG pathway classification of Vero cells showing the number of gene types in specific pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003e Volcano plot of Vero cells showing the statistically significant difference in gene expression levels.\u003c/p\u003e\n\u003cp\u003eComparison Group: Microgravity/ Normal gravity, Significant difference threshold: |log2FC| \u0026gt;= 1, Qvalue \u0026lt;= 0.05.\u003c/p\u003e\n\u003cp\u003eThe arrow indicates the specific genes mentioned in the Discussion.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/4ec64e5a3d6c3b23d6523b34.jpg"},{"id":94984666,"identity":"815e4037-9d54-4007-aa7f-514767035bec","added_by":"auto","created_at":"2025-11-03 06:54:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1186221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/8b4f99ae-e27d-4e06-84ec-0a37e1a2bd95.pdf"},{"id":94756511,"identity":"fba33d9a-0836-494b-acaa-e224f1c3f6e3","added_by":"auto","created_at":"2025-10-30 11:17:32","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27652,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/7640d332fa218a09aa51b857.xlsx"},{"id":94823698,"identity":"b8cad911-5062-41e3-bdaf-70b36a9ca8f2","added_by":"auto","created_at":"2025-10-31 06:47:51","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8343990,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure1a.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/e3684fd992738282b80e866d.mp4"},{"id":94824039,"identity":"cf67e647-ce1b-4a67-872d-37efdee30806","added_by":"auto","created_at":"2025-10-31 06:48:23","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":70630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Microgravity simulator\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Movie of rotating clinostat (Advanced Engineering Services co., Ltd.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e iP-TEC® Flasks with a vented cap.\u003c/p\u003e","description":"","filename":"Slide6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6810961/v1/e855e1272b54c82d58de8145.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Observation of host cell morphological changes under simulated-microgravity conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs space development progresses, more and more people are expected to stay in space in the future. However, humans in space experience greater stressors compared to those on Earth, primarily due to factors like microgravity, radiation exposure, and confinement within a closed environment. These stressors contribute to muscle and bone loss\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, tissue inflammation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, central nervous system disorders\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and various other health risks. Furthermore, some astronauts have developed opportunistic infections with viruses such as cytomegalovirus\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e or Herpes virus\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e during their stay on the International Space Station (ISS) due to decreased immune function. In fact, there have been 29 reported infections among a total of 742 crew members in the space shuttle program so far\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While detailed studies of infection in microgravity are necessary to explore various opportunistic pathogens, there is limited knowledge about how these pathogens behave within the host cells. To date, animal cell culture microgravity-simulation studies have been conducted on only a limited number of cell types such as stem cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, skin cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and cancer cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. An in-depth understanding of the effects of microgravity on host cells is not only needed for future research on infectious diseases, but also to help protect the health of astronauts.\u003c/p\u003e\u003cp\u003eThis study focuses on two types of host cells, Vero cells and human foreskin fibroblasts (HFFs), which can serve as models of host response during infection with opportunistic pathogens and observe their behavior under conditions that simulate a microgravity environment. Vero cells are cultured cells derived from the kidneys of an African green monkey and have been widely employed in global microbiology research, pathogen testing in medical settings, and viral vaccine production\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Vero cells lack interferon production and are susceptible to a wide range of viruses; however, due to this limitation, they cannot adequately represent human cells in terms of immune response to pathogens. Therefore, it is essential to compare findings from Vero cells with those from primary cell lines such as HFFs. Herpes virus, a concern for astronaut infections, has been cultured in both Vero cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and HFFs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, these cell types were selected to study viral infections in microgravity.\u003c/p\u003e\u003cp\u003eWe create a simulated microgravitational environment by using a 3D clinostat (Advanced Engineering Services co., ltd.). This device rotates a flask filled with medium, and the gravity on cells is distributed. It has been utilized in multiple studies, and its effectiveness has been demonstrated\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For example, skin cells cultured in a 3D clinostat exhibit a reduction in collagen, like the skin cells of astronauts after a space mission\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Compared to the microgravity experiments conducted on the ISS or in aircraft, it is possible to create a stable environment inexpensively with a 3D clinostat.\u003c/p\u003e\u003cp\u003eOur study examined the effects of simulated microgravity by analyzing the number, morphology, and variation in expression genes of host cells. Our findings will lead to further investigation into the effects of microgravity on host cells, to analyze the causes of opportunistic infections.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eCells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVero cells (provided by Dr. Y. Kawaguchi) were maintained in Dulbecco Minimal Essential Medium (DMEM: Nacalai Tesque) supplemented with 5% or 2% fetal bovine serum (FBS) and penicillin (100 U/ml) -streptomycin (100 \u0026micro;g/ml) solution (Nacalai Tesque). Human foreskin fibroblasts (HFFs) (ATCC: SCRC-1041) were maintained in RPMI 1640 medium (Nacalai Tesque) supplemented with 5% or 2% fetal bovine serum (FBS) and penicillin (100 U/ml) -streptomycin (100 \u0026micro;g/ml) solution (Nacalai Tesque). The cells are cultured in 5% FBS until the flask reaches 80% confluent. They are maintained in a 2% FBS environment under simulated microgravity environment. All cultures were maintained at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSimulated microgravity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were subjected to a microgravitational environment by use of a 3D clinostat (Advanced Engineering Services co., ltd.) (Supplementary Fig.\u0026nbsp;1a). They rotated in an incubator kept at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The average gravitational force acting on the cells was reduced to about 10^-3 when the clinostat rotated at 30 rpm\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe used iP-TEC\u0026reg; Flasks (25-cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) with a vent cap (Supplementary Fig.\u0026nbsp;1b) to rotate the samples in the clinostat. These flasks have a semi-permeable cap and were fully filled with medium during the experiments. The flasks were fixed to the clinostat with a band. Several flasks could be kept in the same incubator so that comparative experiments could be also performed.\u003c/p\u003e\u003cp\u003eIn this research, every sample was first cultured in normal gravity for 2 days. Then, the cells were transferred to the 3D clinostat to start the culture (day 0); at this point, they were 80% confluent. The cells were cultured in simulated microgravity for 7 days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eElectron Microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVero cells (cultured under normal gravity and simulated microgravity for 7 days) were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4, 4\u0026deg;C, then gently removed from the flasks by using a cell scraper and pelleted via centrifugation (1000rpm, 5min). The pellets were thoroughly rinsed with 0.1 M PB. Then the pellets were fixed with 1% OsO\u003csub\u003e4\u003c/sub\u003e in 0.1 M PB (pH 7.4) for 2 h at 4\u0026deg;C. The fixed pellets were then rinsed with 70% ethanol, dehydrated through a graded ethanol series and propylene oxide, and embedded in Epon 812.\u003c/p\u003e\u003cp\u003eUltrathin sections (80-nm-thick) were cut using an EM UC7i ultramicrotome (Leica microsystems) and examined using an HT7700 transmission electron microscope (Hitachi) without uranyl acetate or lead citrate staining. The brightness and contrast of the obtained TIFF data were adjusted by using Photoshop CS6 (Adobe).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA sequencing analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVero cell (cultured under normal gravity and simulated microgravity for 7 days) mRNA was purified by using a FastGene\u0026trade; RNA Premium Kit (50 preps: Nippon Genetics) for RNA sequencing (Transcriptome). RNA sequencing was performed by BGI Japan. Paired-end libraries were sequenced using the DNBSEQ- (2 \u0026times; 150 nucleotide read length). platform, which generated about 6.73G Gb bases per sample. The average mapping ratio with the reference genome was 97.68%, and the average mapping ratio with the reference gene was 77.32%. Bioinformatics analyses were performed using BGI's proprietary Dr. TOM software and BGI's in-house customized data mining system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biosys.bgi.com\u003c/span\u003e\u003cspan address=\"https://biosys.bgi.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStaining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSample cells were stained with 50 \u0026micro;g/ml propidium iodide (PI: Nacalai Tesque) solution (Absorbance: 40-fold dilution, water λ max: around 492 nm). Dead cells fluoresced red when incubated at room temperature for 5 minutes in the dark.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStudent's t-test was performed for comparisons between two normally distributed samples, using the statistical analysis function in Microsoft Excel. Differences between the experimental groups were considered significant at *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE of the mean (SEM).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIn a simulated-microgravity environment, Vero cells form three-dimensional aggregates.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe cultured Vero cells in normal gravity and in the simulated microgravity for 7 days. In the microgravity environment, Vero cells formed several three-dimensional aggregates known as multicellular spheroids (MCSs)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In contrast, Vero cells cultured under normal gravity maintained a sheet-like morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Higher magnification images of these cells on day 7 further detailed their respective morphologies under both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To examine changes immediately after transfer to microgravity, we observed Vero cells every 2 h after they were placed in the simulated-microgravity environment. We observed that the MCSs formed rapidly, within 4 to 6 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Magnified views of the cells at the 4h and 6h time points illustrate this aggregation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). We then compared Vero cells cultured under simulated microgravity for 24 h with those cultured under simulated microgravity for 24 h followed by normal gravity conditions for another 24 h. The results revealed that the formation of the MCSs was irreversible (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Of note, these MCSs primarily consisted of live cells, as evidenced by partial staining with the PI solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhile our analysis primarily focused on Vero cells as a representative cell line, it is imperative to investigate the effects of simulated microgravity on primary cells to ascertain whether similar phenotypic changes occur. Consequently, we conducted analogous experiments with human foreskin fibroblasts (HFFs). After 7 days of culture, HFFs exhibited structures resembling MCSs, although smaller than those observed with Vero cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Unlike the Vero cell MCSs, which appeared to consist of tens of cells, the HFF MCSs comprised only 2\u0026ndash;3 cells based on optical microscopy observations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther analysis using transmission electron microscopy (TEM) revealed changes in cell thickness and intercellular spacing for both Vero cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and HFFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSimulated microgravity promotes cell proliferation.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe number of cells after 7 days of culture was measured for flasks that initially contained the same number of cells. A significant increase in the number of Vero cells under simulated microgravity, with an approximately 1.6-fold difference in cell numbers, was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor HFFs, the number of cells was compared between static culture and microgravity after 7 days of culture. HFFs also showed an approximately 1.2-fold increase in cell numbers, although statistical significance was not confirmed by t-test results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eIn addition to static culture and simulated microgravity, to investigate the effects of cell directions and gravity, we prepared flasks in upside down and sideways condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Sheet-like cultured cells were cultivated for 7 days under four conditions: normal gravity standing, normal gravity sideways, normal gravity upside-down, and simulated microgravity. MCS formation and increased cell numbers were observed only under simulated microgravity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges in gene expression levels.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine how microgravity affects gene expression, a comparative analysis of mRNA expression in cells cultured under normal gravity and microgravity was performed. Gene Ontology (GO) Classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), KEGG pathway classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), and a Volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) of the results are shown. In cells cultured under microgravity, 18 Differentially Expressed Genes (DEGs) were upregulated and 106 DEGs were downregulated (Q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 log2FC\u0026thinsp;\u0026gt;\u0026thinsp;1) compared to cells cultured under normal gravity (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the GO classification, the X-axis represents the number of genes annotated to GO terms, and the Y-axis represents the category of GO terms. Each bar shows the number of genes with similar functions. In the KEGG pathway classification, performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, the vertical axis is the number of genes annotated to a category of the KEGG Pathway, and the horizontal axis is the category of KEGG Pathway. In the volcano plot, the vertical axis represents the statistical significance of the data, whereas the horizontal axis indicates the expression ratio. Red represents upregulated DEGs, green represents downregulated DEGs, and gray represents non-DEGs.\u003c/p\u003e\u003cp\u003eFrom the result of GO classification, we identified 14 DEGs involved in cell proliferation. We observed two patterns: an increase in cell proliferation-promoting factors or a decrease in cell proliferation-inhibiting factors, and conversely, a decrease in cell proliferation-promoting factors or an increase in cell proliferation-inhibiting factors. Of the 14 DEGS, the five that showed the most significant changes in expression level (i.e., \u003cem\u003eADM, HILPDA, STC1, BAMBI\u003c/em\u003e, and \u003cem\u003eIL6\u003c/em\u003e) were decreases in cell proliferation-promoting factors\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We also obtained 15 DEGs involved in the immune system. Of these genes, the five most noteworthy, which all decreased, were \u003cem\u003eLOX, CCN4, IL6\u003c/em\u003e, and \u003cem\u003eBNIP3\u003c/em\u003e), which are involved in the formation and survival of malignant tumors, and \u003cem\u003ePTX3\u003c/em\u003e, which is involved in humoral innate immunity\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found that simulated microgravity has an impact on the morphology and proliferation of cells. Vero cells showed proliferation with statistical significance, and for the HFFs, the t-test yielded a \u003cem\u003eP\u003c/em\u003e-value of approximately 0.13, suggesting potential variability in the values. Previous research has suggested that alterations in the cytoskeleton and extracellular matrix may cause MCS formation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our electron microscopy findings support this hypothesis. As the MCSs increase over time, it suggests that MCSs are formed through the stacking of divided cells in a spherical configuration.\u003c/p\u003e\u003cp\u003eHowever, the increased Vero cell numbers cannot be solely attributed to MCS formation due to their sparse density. Insights into the increase in cell numbers can be gleaned from the electron microscopy images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Cells observed under the electron microscope appeared detached and suspended from the flask surface. Therefore, it is likely that the images depict cells in a sheet-like rather than MCS formation, as indicated by the high density of stacked cells, allowing more cells to be accommodated to restricted space. Similar reasoning applies to the HFF electron microscopy images, supporting the notion that simulated microgravity induces three-dimensional structural changes in sheet of cells.\u003c/p\u003e\u003cp\u003eBased on our results from the four conditions tested (i.e., normal gravity standing, normal gravity sideways, normal gravity upside-down, and simulated microgravity), we infer that simulated microgravity induces an effect that does not merely reflect a change the direction of gravity acting on cells.\u003c/p\u003e\u003cp\u003eThe RNA sequencing results from Vero cells revealed differences in gene expression between static cultivation and simulated-microgravity conditions. The GO classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) indicated a decrease in cell proliferation factors, and we were unable to obtain evidence that cell proliferation was genetically promoted. In contrast, the KEGG pathway Classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) revealed a significant decrease in the expression of \u003cem\u003eTNFRSF10B\u003c/em\u003e, which is part of the apoptosis-inducing pathway\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e within the \u0026ldquo;cell growth and death\u0026rdquo; category. This could potentially inhibit apoptosis, thereby leading to an increase in cell numbers.\u003c/p\u003e\u003cp\u003eFrom the GO classification, the expression of two prominent genes (\u003cem\u003eCXCR4\u003c/em\u003e, and \u003cem\u003eNDRG1\u003c/em\u003e) associated with cell junctions was found to be downregulated. The upregulation in \u003cem\u003eCLDN2\u003c/em\u003e may be an adaptive response to weakened cell adhesion. Although not included among the genes associated with cell junctions, we also observed an decrease in the expression of growth differentiation factors (e.g., \u003cem\u003eGDF11\u003c/em\u003e) that are involved in collagen secretion. Our results suggest a potential weakening of cell adhesion and adaptive responses, consistent with previously reported reductions in collagen levels\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The statistically significant decrease in the expression of the exocytosis-enhancing factor (\u003cem\u003eNDRG1\u003c/em\u003e) also indicates a possible link to the decline in immune function under microgravity conditions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdditionally, we examined other genes that showed notable differences in expression levels in a volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The most upregulated gene, \u003cem\u003eTMEM151A\u003c/em\u003e, plays a role in paroxysmal movement disorders\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, but its relevance here is unclear. \u003cem\u003eNAT8\u003c/em\u003e and \u003cem\u003eISM1\u003c/em\u003e, which were the second and third most upregulated genes, respectively, have been reported to assist in viral replication\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and to modulate antiviral activity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, suggesting a potential involvement in immune responses.\u003c/p\u003e\u003cp\u003eIn summary, in this study, we detected changes in host cells under simulated-microgravity conditions through morphological observations, cell count measurements, and RNA sequencing. Our findings from electron microscopy suggest that these cellular changes may be influenced by not only endocrine factors but also physical forces. The implications of our findings for pathogen growth remain unclear. Nonetheless, it is essential to consider such changes in host cells when investigating infectious diseases under microgravity conditions. The findings from this study provide a novel perspective on opportunistic infections during space missions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eAll authors declare no financial competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eThis research was supported by a Grant-in-Aid for Scientific Research (B: 24K01921) from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan, by a Livestock Promotional Subsidy from the Japan Racing Association, and by Front Loading Research from the Advisory Committee for Space Utilization Research in the Japan Aerospace Exploration Agency (JAXA) and the Institute of Space and Astronautical Science (ISAS).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.Y. designed the study, collected data and drafted the manuscript. H.B. and D.K. contributed to collecting data and helped draft the manuscript. F.M. contributed to data analysis and edited the manuscript. K.K. supervised the project and edited the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analysed during the current study are available in the Sequence Read Archive (SRA) repository, under the BioProject accession number PRJNA1283931 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1283931).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNagaraja, M. P. \u0026amp; Risin, D. The current state of bone loss research: data from spaceflight and microgravity simulators. \u003cem\u003eJ. Cell. Biochem.\u003c/em\u003e \u003cb\u003e114\u003c/b\u003e, 1001\u0026ndash;1008 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlaber, E., Mar\u0026ccedil;al, H. \u0026amp; Burns, B. P. Bioastronautics: the influence of microgravity on astronaut health. \u003cem\u003eAstrobiology\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 463\u0026ndash;473 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel, Z. S. et al. Red risks for a journey to the red planet: The highest priority human health risks for a mission to Mars. \u003cem\u003enpj Microgravity\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e (1), 33 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKokhan, V. S., Shakhbazian, E. V. \u0026amp; Markova, N. A. Psycho-emotional status but not cognition is changed under the combined effect of ionizing radiations at doses related to deep space missions. \u003cem\u003eBehav. Brain. Res.\u003c/em\u003e \u003cb\u003e362\u003c/b\u003e, 311\u0026ndash;318 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMehta, S. K. et al. Reactivation and shedding of cytomegalovirus in astronauts during spaceflight. \u003cem\u003eThe J. Infect. diseases\u003c/em\u003e. \u003cb\u003e182\u003c/b\u003e, 6 1761\u0026ndash;1764 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRooney, B. V. et al. Herpes virus reactivation in astronauts during spaceflight and its application on earth. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 432964 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrucian, B., Kunz, H. \u0026amp; Clarence, F. Sams. Risk of crew adverse health event due to altered immune response. No. JSC-CN-33468. (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePisanu, M. et al. Lung cancer stem cell lose their stemness default state after exposure to microgravity. \u003cem\u003eBioMed Res. international\u003c/em\u003e \u003cb\u003e2014\u003c/b\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi, D. et al. 3D cell culture using a clinostat reproduces microgravity-induced skin changes. \u003cem\u003enpj Microgravity\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e (1), 20 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhn, J. et al. Morphologic and genomic changes of thyroid cancer cell lines exposed to conditions of simulated microgravity. \u003cem\u003enpj Microgravity\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e (1), 8 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSheets, R. History and characterization of the Vero cell line. \u003cem\u003eCent Biol. Eval Res\u003c/em\u003e \u003cb\u003e1\u0026ndash;12\u003c/b\u003e (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen, C. F. et al. Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus‐1. \u003cem\u003eBiotechnol. J.\u003c/em\u003e \u003cb\u003e19.1\u003c/b\u003e, 2300244 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRasmussen, L. U. C. Y. Farley. Inhibition of Herpesvirus hominis replication by human interferon. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cb\u003e12.1\u003c/b\u003e, 104\u0026ndash;108 (1975).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, Y. et al. Time-averaged simulated microgravity (taSMG) inhibits proliferation of lymphoma cells, L-540 and HDLM-2, using a 3D clinostat. \u003cem\u003eBiomed. Eng. Online\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 1\u0026ndash;12 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi, D. et al. 3D cell culture using a clinostat reproduces microgravity-induced skin changes. \u003cem\u003enpj Microgravity\u003c/em\u003e 7.1 20 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanehisa, M. \u0026amp; Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 27\u0026ndash;30 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanehisa, M., Furumichi, M., Sato, Y., Kawashima, M. \u0026amp; Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e, D587\u0026ndash;D592 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen et al. Adrenomedullin promotes human endothelial cell proliferation via HIF-1α. Molecular and cellular biochemistry 365 : 263\u0026ndash;273. (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSheng, Y. et al. Hypoxia-inducible lipid droplet-associated (HILPDA) facilitates the malignant phenotype of lung adenocarcinoma cells in vitro through modulating cell cycle pathways. \u003cem\u003eTissue Cell.\u003c/em\u003e \u003cb\u003e70\u003c/b\u003e, 101495 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBai, Y. et al. Stanniocalcin 1 promotes cell proliferation via cyclin E1/cyclin\u0026ndash;dependent kinase 2 in human prostate carcinoma. \u003cem\u003eOncol. Rep.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e (4), 2465\u0026ndash;2471 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou, L. et al. The overexpression of BAMBI and its involvement in the growth and invasion of human osteosarcoma cells. \u003cem\u003eOncol. Rep.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (3), 1315\u0026ndash;1322 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOgata, A. et al. IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. \u003cem\u003eJournal of immunology\u003c/em\u003e (Baltimore, Md.: 159.5 (1997): 2212\u0026ndash;2221. (1950).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTenti, P. \u0026amp; Vannucci, L. Lysyl oxidases: linking structures and immunity in the tumor microenvironment. \u003cem\u003eCancer Immunol. Immunother.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e (2), 223\u0026ndash;235 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh, K. \u0026amp; Sunday, S. Oladipupo. An overview of CCN4 (WISP1) role in human diseases. \u003cem\u003eJ. Translational Med.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (1), 1\u0026ndash;24 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNeurath, M. F. \u0026amp; Susetta Finotto IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer. \u003cem\u003eCytokine Growth Factor Rev.\u003c/em\u003e \u003cb\u003e22.2\u003c/b\u003e, 83\u0026ndash;89 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNey, P. A. Mitochondrial autophagy: Origins, significance, and role of BNIP3 and NIX. \u003cem\u003eBiochim. et Biophys. Acta (BBA)-Molecular Cell. Res.\u003c/em\u003e \u003cb\u003e1853\u003c/b\u003e (10), 2775\u0026ndash;2783 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeban, L. et al. Pentraxins in innate immunity: lessons from PTX3. \u003cem\u003eCell Tissue Res.\u003c/em\u003e \u003cb\u003e343\u003c/b\u003e (1), 237\u0026ndash;249 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, A., Wilson, N. S. \u0026amp; Ashkenazi, A. Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation. \u003cem\u003eCurr. Opin. Cell Biol.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (6), 837\u0026ndash;844 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHodgson, R. et al. NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance. \u003cem\u003eCommun. Biology\u003c/em\u003e. \u003cb\u003e5.1\u003c/b\u003e, 1216 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnna, M. et al. Systems approach reveals distinct and shared signaling networks of the four PGE2 receptors in T cells. \u003cem\u003eSci. Signal.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, eabc8579 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, H. F. et al. TMEM151A variants cause paroxysmal kinesigenic dyskinesia. \u003cem\u003eCell. Discovery\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e (1), 83 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao, X. et al. N-Acetyltransferase 8 promotes viral replication by increasing the stability of enterovirus 71 \u003cem\u003enonstructural proteins\u003c/em\u003e. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e, e00119\u0026ndash;e00122 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, C. et al. Zebrafish Ism1 is a novel antiviral factor that positively regulates antiviral immune responses. \u003cem\u003eDev. Comp. Immunol.\u003c/em\u003e \u003cb\u003e125\u003c/b\u003e, 104210 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6810961/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6810961/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrogravity exposure has been associated with various health concerns, including reports of opportunistic infections. However, the specific changes that occur in host cells under these conditions remain unclear. In this study, we investigated the effect of microgravity on Vero cells and human foreskin fibroblasts (HFFs), which serve as host cells.\u003c/p\u003e\u003cp\u003eUnder simulated-microgravity conditions, both Vero cells and HFFs formed multicellular spheroids, representing an irreversible change to the viable cells. Moreover, Vero cell proliferation after 7 days of culture under simulated-microgravity conditions was increased compared to normal gravity conditions. Electron microscopy revealed widened intercellular spaces under the simulated-microgravity conditions, indicating morphological changes. RNA sequencing showed no substantial changes in the expression levels of genes related to growth ability. However, some changes in gene expression support the phenomena like reductions in collagen levels, which are reported previously. Our findings suggest that simulated microgravity influences cell morphology and proliferation, and offer insights into maintaining the health of astronauts.\u003c/p\u003e","manuscriptTitle":"Observation of host cell morphological changes under simulated-microgravity conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 11:17:27","doi":"10.21203/rs.3.rs-6810961/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-14T03:25:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266192107239050505833187277120530700995","date":"2026-03-25T21:16:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79506029080101578045577705519012566329","date":"2026-03-24T07:21:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203964749564338081016571229794475743112","date":"2025-10-21T11:47:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-16T10:16:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T12:00:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-11T20:08:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-02T09:24:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-02T09:20:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"832a3fa5-1232-4f31-8e43-faa6b5693669","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56886844,"name":"Biological sciences/Cell biology/Cell growth"},{"id":56886845,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2025-10-30T11:17:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 11:17:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6810961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6810961","identity":"rs-6810961","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}