不同防冻剂对兔胚胎干细胞慢速冷冻保存的影响
The Effects of Different Cryoprotectants on the Cryo preservation of Rabbit Embryonic Stem Cells
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摘要: 干细胞冷冻保存是干细胞研究和临床应用中的必需技术。为提高兔胚胎干细胞在慢速冻存过程中的保存效果，比较了二甲基亚砜(DMSO)和乙二醇(ethylene glycol，EG)对兔胚胎干细胞冷冻保护效果。对冷冻复苏后的细胞进行台盼蓝染色，并研究其胚胎干细胞分子特性，结果表明DMSO比EG具有更好的冷冻保护效果。再在以10% DMSO为基础的防冻液中添加膜稳定剂海藻糖(trehalose)或谷氨酰胺(glutamine)，细胞冷冻复苏后结果显示，谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用，使细胞存活率从71%提高到83.7%。当谷氨酰胺浓度为0、5、10、20、40 mmol/L分别加入防冻液中后，20 mmol/L的谷氨酰胺具有最佳的冷冻保护效果。以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为：在胚胎干细胞培养液中添加10% DMSO＋20 mmol/L谷氨酰胺。Abstract: An effective freezing-thawing technique is crucial for the clinical application of newly derived rabbit embryonic stem (ES) cells. The aim of this study was try to find an optimal cryopreservation protocol for rabbit embryonic stem cells using slow freezing-rapid thawing without a programmable freezer. We tested the effects of the following cryoprotective agents (CPAs) on the post-thaw survival, proliferation and differentiation capacity of rabbit embryonic stem cells: ethylene glycol (EG), dimethyl sulphoxide (Me2SO, DMSO), trehalose and glutamine. Trypan blue exclusion tests showed that, among the CPA treatments in this study, EG was more toxic to rabbit embryonic stem cells than DMSO. The highest survival rate (83.7%) was obtained when the rabbit embryonic stem cells was cryopreserved with 10%DMSO and 20 mmol/L glutamine in the ES cell culture media. Thawed ES cells kept their pluripotency and differentiation potential.
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