Study On The Culture And Freezing Of Mouse Spermatogonial Stem Cells In Vitro

In order to gain more efficient method of how to isolate, culture and frozen Spermatogonial Stem Cells (SSCs), and provide more clinical experience for curing the male infertility and conserving the endangered species, the isolation, purification, culture and cryopreservation of mouse SSCs obtained from the testis of new born mouse (7 days) were studied. Especially, the efficient culture can lay the foundation for the transplantation, pluripotent differentiation, transgenic and in vitro induction, which has potential value on the medical research, biological research and biotechnology.1. 4 cell-chains, 8 cell-chains and 16 cell-chains were the typical conditions of mouse SSCs, which could be observed through the morphology. Alkaline Phosphatase (AKP) existed on SSCs, NBT/BCIP kit was used to identify them by staining. The SSCs was Blue-purple or brown under the Inverted Microscope by AKP staining. Beta-actin and Ngn3 genes were the unique genes, so they can be used to identify the SSCs by RNA isolation and RT-PCR.2. EGF, IGF-1and bFGF were utilized to culture the SSCs by single factor, the double factors and the triple factors. The results showed that the growth rate of EGF (10ng/ml) based on the pre-treatment with 10% FSH (30 ng/ml) was 41.60% (p<0.05); the growth rate of EGF (20ng/ml) and IGF-1(20ng/ml) combination based on post-treatment with 10% FSH (30 ng/ml) was 63.00%(p<0.05); the growth rate of EGF(20ng/ml), IGF-1(20ng/ml) and bFGF (10ng/ml) combination based on post-treatment with 10% FSH(30 ng/ml) was 56.60% (p<0.05).3. LDL, trehalose and lecithin were used in cryopreservation of mouse SSCs by single one and combinations. The results showed the recovery rates of the 1mg/ml LDL group, 3 mg/ml LDL and 0.5 mg/ml lecithin combination were 87.16%(p<0.05) and 90.23%(p<0.05) respectively, which were the best results..4. LDL, trehalose and lecithin were firstly used as in cryopreservation of mouse Sertoli cells and the results were effective. The recovery rates were checked out at the first, seventh and fifteenth day.The results showed that the recovery rates were greatest at the seventh day. The recovery rates on LDL, trehalose and lecithin group were 70.65% (p<0.05), 72.86% (p<0.05), 68.25% (p<0.05) at the seventh day respectively.