Assistant Professor University of Kentucky, United States
Introduction: : Space travel affects multiple organ systems and is linked to decreased bone density, muscle wasting, and cardiovascular changes from space hazards like microgravity [1]; however, much remains unknown regarding the effects of microgravity on most organ systems. Microgravity research on organ systems, especially the female reproductive system, is necessary for safe space exploration, especially as mission lengths increase. Current microgravity research is limited, with most studies using animal cells and 2D cell culture models, and largely focusing on ovarian cells, which show decreased viability, morphological changes, and defective maturation after simulated microgravity [2-4]. Research on uterine, vaginal, and placental cell types in microgravity is critical for the menstrual and reproductive health of astronauts, and necessary for potential human colonization of space. The use of 3D cell models offers more physiologically relevant insight into the effects of microgravity, compared to the more common 2D models. GelMA hydrogels have been used to model various aspects of female reproductive health and can provide a platform to model different reproductive tissues in microgravity [5]. The project objective is to develop a pipeline to evaluate microgravity effects on 3D reproductive cell models so we can begin to understand the effects of space conditions on female reproductive health.
Materials and
Methods: : As previously described [5], we synthesized and characterized a library of gelatin methacryolyl (GelMA) hydrogels (5 wt%) from porcine gelatin Type A (Sigma). We encapsulated various reproductive cells (Vaginal Epithelial Cells, LifeLine Cell Technology; Human Endometrial Endothelial Cells; Innoprot; hTERT-immortalized human endometrial stromal cells; ATCC) of varying cell densities (100,000-500,000 cells/mL) within the hydrogels. To simulate microgravity, we cultured these hydrogels for up to 3 days in a rotating cell culture system previously developed by NASA (RCSS; Synthecon) to replicate aspects of low gravity that allow for investigation into space-like environments on Earth. Subsequently, we evaluated microgravity-induced changes in the cells, including viability, morphology, proliferation, and expression of relevant factors, compared to control samples. This methods pipeline was optimized to focus on culture time, rotation speed, sample size, and overall engineering workflow.
Results, Conclusions, and Discussions:: Our objective of this project was to develop a pipeline using 3D GelMA models to evaluate microgravity effects on reproductive health. We successfully developed and optimized a pipeline to investigate the effects of microgravity on human reproductive cells (Figure 1a). Based on preliminary studies, we observed a slight decrease in cell viability of human vaginal epithelial cells in microgravity, with 51% viable cells compared to 57.5% in static culture (Figure 1b, c). These results are similar to previous studies that show decreased viability of mouse granulosa cells in microgravity compared to static controls [3]. Repeated experiments and larger sample sizes are needed to confirm results due to some challenges related to the experimental pipeline. This pipeline addresses an important gap in the field, which lacks models that replicate the 3D architecture of various reproductive tissues. Ongoing work includes the culture of endometrial microvascular and stromal cells in microgravity to investigate space effects on angiogenesis and decidualization. In conclusion, this work establishes a pipeline that can be used in future and ongoing studies with additional reproductive cell types, allowing for investigation into the effect of microgravity on reproduction.
Acknowledgements and/or References (Optional): : This research is supported by the National Science Foundation REU Program, University of Kentucky (2150337), UK Pigman College of Engineering, UK Dept. of Mechanical & Aerospace Engineering, The Lighthouse Beacon Foundation, UK Materials Research Priority Area, and UK UNITE Research Priority Area.
References:
[1] B. Mishra and U. Luderer, "Reproductive hazards of space travel in women and men," (in English), Nat Rev Endocrinol, vol. 15, no. 12, pp. 713-730, Dec 2019, doi: 10.1038/s41574-019-0267-6.
[2] C. Wu et al., "Simulated microgravity compromises mouse oocyte maturation by disrupting meiotic spindle organization and inducing cytoplasmic blebbing," PLoS One, vol. 6, no. 7, p. e22214, 2011, doi: 10.1371/journal.pone.0022214.
[3] S. Zhang et al., "Simulated Microgravity Using a Rotary Culture System Compromises the In Vitro Development of Mouse Preantral Follicles," PLoS One, vol. 11, no. 3, p. e0151062, 2016, doi: 10.1371/journal.pone.0151062.
[4] H. J. Cho et al., "Microgravity inhibits decidualization via decreasing Akt activity and FOXO3a expression in human endometrial stromal cells," Sci Rep, vol. 9, no. 1, p. 12094, Aug 20 2019, doi: 10.1038/s41598-019-48580-9.
[5] S. G. Zambuto, K. B. H. Clancy, and B. A. C. Harley, "A gelatin hydrogel to study endometrial angiogenesis and trophoblast invasion," (in English), Interface Focus, vol. 9, no. 5, Oct 6 2019, doi: ARTN 20190016 10.1098/rsfs.2019.0016.
