Microgravity significantly impacts human physiology during spaceflight, affecting bone and muscle mass, brain structures, and fluid distribution. Skin is particularly affected, with skin rashes, hypersensitivity, dermatitis, peeling, and infection frequently reported among astronauts. Skin thinning and loss of elasticity are also observed post-flight, as demonstrated in studies of both astronauts and mice exposed to prolonged microgravity. These changes may be linked to altered angiogenesis and collagen turnover. Due to the cost and difficulty of spaceflight studies, clinostats are used to simulate microgravity environments. This research utilizes a previously developed 3D clinostat, generating a time-averaged simulated microgravity (taSMG) condition, to investigate the effects of microgravity on skin in a 3D in vitro setting, offering a more physiologically relevant model than 2D cultures. Previous studies primarily focused on 2D cultures, lacking the complex cellular interactions and structural properties of 3D models, which better reflect in vivo conditions. This study aimed to use a 3D clinostat to simulate microgravity and observe changes in a 3D in vitro skin model composed of keratinocytes, fibroblasts, and endothelial cells.

Numerous studies have documented the adverse effects of microgravity on various physiological systems. Skin-related issues are frequently reported by astronauts, including rashes, irritation, and thinning. Animal studies, such as the one involving mice on the ISS, have shown dermal atrophy and altered collagen turnover in response to prolonged microgravity. Research on endothelial cells has also indicated that microgravity influences angiogenesis, potentially contributing to skin irritation and inflammatory conditions. Clinostats, which simulate microgravity by constantly changing the direction of the gravity vector, have been employed in various studies to overcome the limitations of conducting experiments in space. However, most previous in vitro studies investigating the effects of simulated microgravity on skin utilized 2D cell culture techniques, limiting the accuracy of mimicking the complex in vivo environment. The use of 3D cell culture techniques, such as spheroids, has emerged as a superior approach to studying cell behavior, as they offer a more realistic representation of tissue architecture and cellular interactions compared to 2D models. This study sought to utilize a validated 3D clinostat to simulate the microgravity environment and examine its effects on skin in a more realistic 3D cell culture model.

The study employed a previously validated 3D clinostat to create a taSMG environment. Three cell types were used: human epidermal keratinocytes (HaCaT), human skin fibroblasts (Hs27), and human umbilical vein endothelial cells (HUVECs). To investigate angiogenesis, HUVECs were cultured on Matrigel in 1G, taSMG, and a 1G-to-taSMG group. For spheroid cultures, mono- and co-cultures (fibroblasts and keratinocytes) were created in ultra-low attachment plates and exposed to either 1G or taSMG for three days. Cell viability was assessed using PrestoBlue. Spheroid thickness and cell size were measured using confocal microscopy and ImageJ software. Immunofluorescence staining was performed to analyze collagen type I and cytokeratin-10 expression. A hydroxyproline assay was used to quantify collagen content. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of genes related to collagen synthesis (COL1A1, MMP-1, P4HA1), connective tissue growth factor (CCN1, CCN2), and cytokeratin-10 (K10). Statistical analysis was performed using Student's t-test, Mann-Whitney U test, and ANOVA.

Under taSMG, the thickness of HUVEC arrangement increased significantly compared to the 1G group (12.92% for 1G-to-taSMG and 59.75% for taSMG). Co-cultured spheroids exhibited a significant decrease in diameter under taSMG (6.66%), with individual keratinocytes also showing reduced size. The α1 chain of type I collagen (COL1A1) was upregulated under taSMG, while connective tissue growth factor (CCN2) was downregulated. The expression of cytokeratin-10 (K10) was significantly increased under taSMG conditions. Hydroxproline assay showed a significant increase in total collagen content at day 7 but not at day 3.

The observed increase in endothelial cell arrangement thickness under taSMG suggests increased angiogenesis, potentially explaining skin irritation reported by astronauts. The decrease in co-cultured spheroid diameter and reduced keratinocyte size mirrors skin thinning observed in astronauts and mice exposed to microgravity. The upregulation of COL1A1 and downregulation of CCN2 are consistent with alterations in extracellular matrix homeostasis observed in microgravity. Increased K10 expression could indicate skin barrier disruption. The results demonstrate that the 3D clinostat successfully reproduced several key physiological and structural changes observed in skin under microgravity conditions.

This study successfully demonstrated that a 3D clinostat can effectively replicate microgravity-induced skin changes in an in vitro setting. The results contribute to a better understanding of the cellular mechanisms underlying these changes and provide a valuable tool for pre-flight research aimed at mitigating the health risks associated with space travel. Future studies could incorporate a wider array of cell types in the 3D model and use organ-on-a-chip technology to further improve the accuracy and longevity of the experimental model.

The co-cultured spheroid model used in this study only included two cell types, and therefore does not fully represent the complexity of real skin. The study only examined the effects of simulated microgravity over a short period, limiting the ability to fully capture the long-term changes observed in astronauts during extended space missions. The use of the immortalized HaCaT cell line, while convenient, might not perfectly reflect the behavior of primary human keratinocytes. Despite these limitations, the study provides a valuable initial step towards understanding the effects of microgravity on the skin.