SD大鼠骨髓间充质干细胞在-80℃低温条件下的冻存

Cryopreservation of bone marrow mesenchymal stem cells from Sprague-Dawley rats at -80℃

背景:以-80℃作为冻存温度条件,探索一种低消耗,简单易行的骨髓间充质干细胞贮存方法。目的:筛选适于骨髓间充质干细胞冻存的最适保护液,并验证骨髓间充质干细胞经较长期低温冻存后的生物学特性。方法:贴壁培养骨髓间充质干细胞,免疫荧光法鉴定骨髓间充质干细胞的生物学特性及纯度;以-80℃为温度条件,不同配比的低糖DMEM培养基、胎牛血清和二甲基亚砜作为冻存保护液,短期冻存骨髓间充质干细胞,选择适于骨髓间充质干细胞冻存的最适保护液;以选择出的保护液冻存骨髓间充质干细胞,分别于1,3,6个月复苏,并继续培养,传代;对传代细胞行免疫荧光鉴定,确定在-80℃下保护液对骨髓间充质干细胞生物学特性的影响。结果与结论:采用80%DMEM+体积分数为10%胎牛血清+10%二甲基亚砜作为冻存保护液,适于在-80℃条件下冻存骨髓间充质干细胞,复苏后的细胞能够在体外增殖,并正常传代,冻存后的骨髓间充质干细胞仍可保持原有的生物学活性。

BACKGROUND： We attempt to explore a low-cost, simple and effective way to cryopreserve bone marrow mesenchymal stem cells at -80 ℃. OBJECTIVE： To screen the optimal cryopreservation fluid for bone marrow mesenchymal stem cells and to verify the biological features of bone marrow mesenchymal stem cells after long-term cryopreservation. METHODS： Bone marrow mesenchymal stem cells were cultured using adherent method and the biological features and purity of cells were detected using immunofluorescence method. Bone marrow mesenchymal stem cells were cryopreserved in the cryoprotectant medium containing low-sugar DMEM, fetal bovine serum and dimethyl sulfoxide at different proportions at -80 ℃ for a short term. Then, the optimal cryoprotectant was selected to storage the bone marrow mesenchymal stem cells. After 1, 3, 6 months of cryopreservation, the cells were resuscitated, cultured and passaged. Passage cells were identified immunofluorescence method to determine the biological features of bone marrow mesenchymal stem cells cryopreserved at -80 ℃. RESULTS AND CONCLUSION： Cryoprotectant medium of 80% DMEM＋10% fetal bovine serum＋10% dimethyl sulfoxide was suitable for cryopreserving MSCs at -80 ℃, and resuscitated cells were able to proliferate in vitro, and passage normally, indicating the cryopreserved bone marrow mesenchymal stem cells still maintain the original biological activity.