A Xeno-free Media for the In Vitro Expansion of Human Spermatogonial Stem Cells

In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS) and bovine serum albumin (BSA). However, the use of animal components during cell culture introduces the risk of contaminating cells with pathogens, and leads to animal epitope expression, rendering them unsuitable for medical use. Therefore, this study set out to develop a xeno-free, fully defined media for the expansion of human SSCs. We show that the molecules Prostaglandin D2 (PGD-2) and Insulin-Like Growth Factor 1 (IGF-1) can replace FBS and BSA in cell culture media without loss of viability or expansion capability, and that Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) inhibitor Y-27632 supplementation improves viability after cryopreservation. Long-term SSC cultures expanded in xeno-free, defined culture conditions shared identical protein expression profiles for well-known SSC markers, while gene expression analyses revealed a significant improvement in quiescent SSC and pan-germ markers. This xeno-free, defined formulation allows for standardized SSC culture free of animal pathogens. ### Competing Interest Statement The authors have a patent pending for the media formulation.