关键词: 冷冻保护剂, 降温速率, 细胞培养, 精原干细胞, 小鼠

Abstract: Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells （SSCs）. Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha (MEMα) supplemented with 10% fetal calf serum (FCS) and 100μg/L glial cell line-derived neurotrophic factor (GDNF). The survived and proliferating SSCs were examined by WST-8 colorimetric assay. Alkaline phosphatase and reverse transcription-polymerase chain reaction (RT-PCR) were performed to confirm if the cultured 96 hours germ cells were still stem cells. Results The best method to cryopreserve SSCs is using cryoprotector containing 10% dimethyl sulfoxide(DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, and the viability of cells in this method is more than 84%; Although the cell viability in nonprogrammed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times.Conclusion The culture condi

Key words: Cryoprotector, Cooling rate, Cell Culture, Spermatogonial stem cell, Mouse