细胞短期冻存液的优化研究

Optimization of Freezing Medium in Short-Period Cell Cryo⁃preservation

目的:探索不同配方的冻存液对细胞短期冻存复苏后存活率及增殖活性的影响,进而对细胞短期冻存液配比进行优化。方法:将多种贴壁细胞及悬浮细胞分别用4种不同配比冻存液冻存[(A)10%二甲基亚砜(DMSO)+10%胎牛血清(FBS)+80%DMEM/1640培养基;(B)10%DMSO+20%FBS+70%DMEM/1640培养基;(C)10%DMSO+50%FBS+40%DMEM/1640培养基;(D)10%DMSO+90%FBS],程序性降温(平均降温速率-1℃/min)至-80℃,冻存4 d后以相同方法复苏细胞并观察细胞生长形态学变化,分别拍摄细胞在复苏后24、30、48、72、96 h时的生长状态,并根据台盼蓝染色进行细胞计数,绘制细胞生长曲线。结果:冻存液中的不同血清浓度配比对贴壁细胞短期冻存复苏后的生长形态及增殖无明显影响;而对于悬浮细胞,高血清配比的冻存液(C、D组)短暂冻存后复苏细胞的存活率明显高于低血清配比组(A、B组),并且其生长增殖能力也明显减弱。结论:对于贴壁细胞的短期冻存与复苏,冻存液中DMEM∶FBS∶DMSO比例为8∶1∶1;而对于悬浮细胞的短期冻存与培养,FBS∶DMSO为9∶1的冻存效果最好。

Objective:To optimize the proportion of short-term cryopreservation solution by exploring the effects of different formulations of cell freezing medium on the survival rate and proliferation activity of cells after short-term cryopreservation and recovery.Methods:Multiple adherent and suspension cell lines were cryopreserved at-80℃for 4 days after programmed cooling(average cooling rate of-1℃/min)using different freezing medium[(A)10%DMSO+10%FBS+80%DMEM/1640;(B)10%DMSO+20%FBS+70%DMEM/1640;(C)10%DMSO+50%FBS+40%DMEM/1640;(D)10%DMSO+90%FBS].The growth status at 24,30,48,72 and 96 h was captured by microscope after recovery by the same method to observe the morphological changes.Cell growth curves were generated according to Trypan-Blue-staining cell count.Results:The concentration of serum in the cell freezing medium had no significant effect on the morphology and viability of adherent cells after short-term cryopreservation and recovery.While for suspension cells,the survival rate of cells which in the high serum ratio(group C and D)was significantly higher than that in the low serum ratio(group A and B),and the growth and proliferation ability were also significantly reduced.Conclusion:For the short-term cryopreservation and recovery of adherent cell lines,DMEM∶FBS∶DMEM/1640=8∶1∶1 was recommended.For suspension cell lines,FBS∶DMSO=9∶1 was recommended to guarantee the viability of cells.