Mouse undifferentiated spermatogonial stem cells cultured as aggregates under simulated microgravity

Dynamic simulated microgravity (SMG) culture systems provide environments that stimulate stem cell proliferation and differentiation. However, the effect of SMG on spermatogonial stem cells (SSCs) remains unclear. Here, we used a rotating cell culture system (RCCS) to determine its effect on mouse SSC proliferation and differentiation. SSCs were enriched from mouse pub testis and cocultured with Sertoli cell feeders pre-treated with mitomycin C on fibrin scaffolds in a rotary bioreactor for 14 days. Our results show that mouse SSCs cultured in a rotary bioreactor exhibited enhanced proliferation surpassing those cultured in static conditions, although SSC cultures in SMG underwent a growth lag at initial 3 days. After 14 days, mouse SSCs and feeders grew into cell aggregates with average diameters of 242.63 ± 16.53 μm compared with those in conventional static culture (49.51 ± 15.64 μm). Related detection revealed that proliferating SSCs in SMG remained undifferentiated, maintained clone-forming capacity and were capable of differentiation into round spermatids with flagella. The growth characteristics of mouse SSCs in RCCS suggest that the resulting aggregates are similar to native in vivo cells. Rotary bioreactors that create SMG environments may be an alternative to conventional systems for the clinical application of SSCs.