人胎脑神经干细胞来源少突胶质前体细胞冻存方法的优化

Optimization of cryopreservative method for human fetal brain neural stem cells-derived oligodendrocyte precursor cells

目的通过改变不同因素条件优化人胎脑神经干细胞来源的少突胶质前体细胞(OPC)冻存方法,进一步提高复苏活率。方法比较使用消化和机械吹打两种方法收集人OPC,采用人多能干细胞(hPSC)无血清冻存液、含930 mL/L胎牛血清(FBS)和70 mL/L二甲基亚砜(DMSO)的冻存液分别冻存细胞,比较两组冻存前后活率。择优后比较4种不同冻存液(含70 mL/L DMSO和930 mL/L FBS的冻存液,含70 mL/L DMSO、300 mL/L FBS的OPC完全培养基,含70 mL/L DMSO、300 mL/L FBS、0.2 mol/mL海藻糖的OPC完全培养基,含70 mL/L DMSO、100 mL/L FBS、300 mL/L羟乙基淀粉溶液的OPC完全培养基)、冻存速率(冻存盒慢速降温和液氮气相快速降温)及冷冻保存时间3个因素对复苏活率的影响。在优选后的冻存方案基础上,复苏后7 d,使用免疫荧光细胞化学染色法比较OPC特异性标志物血小板源性生长因子受体α(PDGFRα)、ST8α-N-乙酰神经酰胺α2,8-唾液酸转移酶1(ST8SIA1/A2B5)、硫酸软骨素蛋白聚糖4(CSPG4/NG2)、增殖相关KI-67抗原(ki67)表达。结果消化组收集细胞,冻存前后活率明显高于机械组;快速降温4组复苏活率均低于30%且无法继续培养,慢速降温4组复苏活率均较高。使用消化法收集细胞,冻存细胞数为2×10^6/mL;使用含海藻糖的冻存液,慢速降温冻存,复苏活率最高;可达(75.73±6.66)%。与对照组相比,复苏后培养组增殖倍数、ki67表达无明显差异,两组均表达PDGFRα、A2B5、NG2。短期和长期储存组复苏活率无明显差别。结论消化液收集OPC,使用含海藻糖的冻存液慢冻快融的方法,适用于人胎脑神经干细胞诱导OPC冷冻保存,复苏后不影响细胞增殖和标志物表达,储存时间对复苏活率无影响。

Objective To explore the impact of various conditions during cryopreservation on the survival rate of oligodendrocyte precursor cells(OPCs)derived from human fetal neural stem cells.Methods We compared the cell viability of oligodendrocyte precursors harvested with or without digestion.Then we tested the impact of 3 factors during cryopreservation,freezing solutions(solution with 70 mL/L DMSO and 930 mL/L FBS;solution with 70 mL/L DMSO,300 mL/L FBS and OPC culture medium;solution with 70 mL/L DMSO,300 mL/L FBS,0.2 mol/mL trehalose and OPC culture medium;solution with 70 mL/L DMSO,300 mL/L FBS,300 mL/L HES and OPC culture medium),freezing methods(the step-wised freezing or rapid freezing within liquid nitrogen)and storage durations for the better survival rate of OPCs.The optimized method with the best survival rate of OPCs was implemented and at day 7 after recovery,the viability,OPCs specific markers[platelet derived growth factor receptor alpha(PDGFRα),ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1(ST8SIA1/A2B5),chondroitin sulfate proteoglycan 4(CSPG4/NG2),ki67 were tested and compared with immunofluorescent cytochemical staining.Results Harvesting with digestion contributed to higher OPCs survival rate.OPCs of rapid freezing had survival rates less than 30%and couldn’t be re-cultured.The step-wised freezing group showed higher recovery rate.Harvesting with digestion,preservation solution with trehalose,using 2.0×10^6/mL of cell number,step-wised freezing,contributed to the highest OPCs survival rate reaching(75.73±6.66)%.Compared with the fresh cultured group,cell proliferation,ki67 antigen,PDGFRα,A2B5 and NG2 expression of OPCs were similar in the recovered cells.Storage duration didn’t affect OPCs survival rate.Conclusion Harvesting with digestion,step-wised freezing,preservation solution with trehalose contribute to higher OPCs survival rate during cryopreservation and cell-thawing.Storage time doesn’t affect phenotypes and viability of OPCs.