[Screening and culture of rat spermatogonial stem cells].

ABSTRACT

AIM: To establish a system of screening and culture of rat spermatogonial stem cells (SSCs). METHODS: We prepared the rat testis cells using the improved two-step enzymatic digestion, and then isolated the rat SSCs by the differential adherence selection method. The highly enriched SSCs were cultured in the serum-free culture medium of DMEM/F12 supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GFRα1 and basic fibroblast growth factor (bFGF) and on rat embryonic fibroblast (REF)feeder layer. The activity of stem cells was examined morphologically and by RT-PCR and immunocytochemical analysis for the SSCs marker gene expressions. RESULTS: The modified two-step enzymatic digestion could effectively isolate the rat testis cells, and from the isolated testis cells, rat SSCs could be successfully purified by the improved method of differential adherence selection. After over-20-day culture of the rat SSCs in serum-free medium, big colonies of SSCs were observed, and the activity of SSCs got affirmed by the positive expressions of SSCs marker genes. CONCLUSION: The system of screening and culture of rat SSCs has been established, which provide a basis for the long-term culture of rat and other animal SSCs.