麦管玻璃化法冷冻保存人诱导多能干细胞

Cryopreservation of human induced pluripotent stem cells(iPS cells) through vitrification in mini straw

目的:麦管玻璃化法冷冻保存人诱导多能干细胞(induced pluripotent cells,iPS细胞)。方法:将机械法切割的iPS细胞团块,依次在10%玻璃化冷冻液和20%玻璃化冷冻液中处理后保存于20%的冷冻液中,封装于0.25 ml麦管并快速置于液氮中保存。解冻时将iPS团块依次置入0.2 mol/L蔗糖溶液和0.1 mol/L蔗糖溶液后,接种至饲养层上培养。检测复苏效率,并对解冻后长期培养的iPS细胞进行特性鉴定,包括:碱性磷酸酶染色、OCT4等免疫荧光染色、RT-PCR法检测内源性oct4和sox2的表达、拟胚体(embryoid,EB)分化、神经细胞分化和畸胎瘤分化能力检测等。结果:麦管玻璃化法冷冻保存的iPS细胞解冻后复苏率可达(77.40±13.12)%,解冻后iPS长期传代培养能维持其特性:碱性磷酸酶阳性,OCT4、SOX2、NANOG、SSEA4、TRA-1-60免疫染色阳性,RT-PCR可检测到内源性oct4和sox2的表达,具备EB、神经细胞和畸胎瘤分化能力。结论:麦管玻璃化冷冻技术可以有效保存iPS细胞,解冻后的iPS细胞在后续传代培养中仍然保持其特性。

Objective：To explore the method of cryopreservating human induced pluripotent.Methods：Colonies of induced pluripotent stem cells（iPS cells） were dissected into pieces（containing approximately 100～200 cells） using mechanical methods,then sequentially treated with 10%,20% vitrification solution,and sealed in mini straws with the second solution.Mini straws were then dropped into liquid nitrogen immediately for vitrification and cryopreservation.After vitrificated preservation,iPS cells were thawed in 37℃ water bath,and were immediately balanced in 0.2 mol/L sucrose solution and then in 0.1 mol/L sucrose solution,and at last plated on feeder layer cells.Alkaline phosphatase activity,expressions of OCT4,SOX2 in iPS cells and cell differentiation were evaluated.Results：Post vitrification and thawing,iPS cells maintain properties of pluripotent stem cells,including normal morphology,alkaline phosphatase staining,OCT4 and SOX2 expression.Calculation of colony recovery rates indicates that approximate（77.40±13.12）% of iPS cell colonies survived the freezing and thaw procedures.Conclusion：Vitrification with ministraws is a very useful and effective cryopreservation method for iPS cells.