The optimized condition for the isolation and in vitro propagation of mouse spermatogonial stem cells

Introduction: The ability for isolation and in vitro propagation of spermatogonial stem cells (SSCs) offer a base for studies on spermatogenesis, and also contribute to the development of new methods for the preservation of livestock and animal genetic modification. The aim of this study was to find the optimal isolation and culture condition for efficient propagation of SSCs.

Methods: Three different isolation methods (mechanical, one-, and two-step enzymatic digestion) were compared to find the optimal isolation method. To find the best culture conditions for in vitro propagation, isolated SSCs were cultured for 7 days in three different culture conditions supplemented with 10% FBS, 0.25% BSA, and 10% KSR, respectively.

Results: The result showed that two-step enzymatic digestion produced a significant high fraction of live cells compared the other two. Non-adhering cells collected after 48 hr and cultured in BSA- and KSR-supplemented medium had a significantly high number of SSCs clump formation compared to FBS-supplemented group. The expression of CD9 confirmed that cell clumps were SSCs clumps. Spermatogonial stem cells cultured in BSA-supplemented medium were positive for NGN3 and PLZF expressions, whereas negative for Stra8 (a meiotic-specific gene) expression, suggesting that most of the cells were undifferentiated SSCs in BSA culture system. In contrast, in FBS- and KSR-supplemented groups, the SSCs were positive forNGN3, PLZF, and Stra8.

Conclusion: These data revealed that two-step enzymatic digestion is the best method for the isolation, and 0.25% BSA-supplemented culture condition is effective for optimal in vitro propagation of SSCs.