不同冻存液对人诱导性多能干细胞冻存与复苏效果的影响

Comparison of the Effects of Different Cryoprotectants on Cryopreservation and Resuscitation of Human Induced Pluripotent Stem Cells

目的:探讨不同冻存液对人诱导性多能干细胞(iPSC)冻存复苏后的存活率及诱导分化的影响,筛选出简单有效的冻存与复苏方法。方法:人iPSC培养至第3代后分组冻存:①含有不同配比胎牛血清(FBS)的细胞冻存液分组:A组[80%培养基(DMEM/F12)+10%FBS+10%二甲亚砜(DMSO)];B组(70%DMEM/F12+20%FBS+10%DMSO);C组(40%DMEM/F12+50%FBS+10%DMSO);D组(90%FBS+10%DMSO)。②含有不同配比DMSO的细胞冻存液分组:E组(45%DMEM/F12+50%FBS+5%DMSO);F组(40%DMEM/F12+50%FBS+10%DMSO);G组(35%DMEM/F12+50%FBS+15%DMSO);H组(30%DMEM/F12+50%FBS+20%DMSO)。以相同方法冻存并复苏人iPSC,观察比较不同组细胞在复苏14 d后重新形成典型干细胞集落的数量,并在相同诱导分化体系下,记录复苏人iPSC向神经元和胶质细胞分化的效率。结果:C和F组细胞复苏后形成典型干细胞集落的数量多于其他组(P<0.05),A和B组细胞复苏后分化为神经元的效率略高于其他实验组。结论:以40%DMEM/F12+50%FBS+10%DMSO配比组合成的细胞冻存液作为人iPSC的冻存保护剂,可使人iPSC的冻存复苏效率最优并保持较好的生物学特性。

ABSTRACT Aim： To select a better-performing cryoprotectant of human induced pluripotent stem cells by comparing the number of the recovery cells and the rate of differentiation in different cryoprotectants through cryopreservation. Methods： Human induced pluripotent stem cells were cultivated to the third generation, and then divided into 8 groups. ①The cell cryopreservation groups with different content serum of cryoprotectants were A, B, C, D. Group A： 80%DMEM/F12＋10%FBS＋10%DMSO; Group B： 70%DMEM/F12＋20%FBS＋10% DMSO; Group C： 40% DMEM/F12＋50%FBS＋10%DMSO; Group D： 90%FBS＋10% DMSO. ②The cell cryopreservation groups with different DMSO of cryoprotectants were E, F, G, H. Group E： 45% DMEM/F12＋50%FBS＋5%DMSO; Group F：40%DMEM/F12＋50%FBS＋10%DMSO; Group G： 35% DMEM/F 12＋50%FBS＋15%DMSO; Group H 30%DMEM/F 12＋50%FBS＋20%DMSO. The survival rate and the rateof differentiation into neurons and astrocytes of the recovery cells among the different groups were compared.Results： Formulation of 40%DMEM/F 12＋50%FBS＋10%DMSO cryoprotectant had the highest cell survivalrate （P〈0.05）. Conclusion： Formulation of 40%DMEM/F 12＋50%FBS＋ 10%DMSO cryoprotectant can makehuman iPS cells cryopreserved optimal efficiently and keep a good biological characteristics.