谭菊 武彩红 文潮潮 王萌

摘要 [目的]建立一种更优化的STO细胞冷冻保存方法。[方法]STO細胞传至第3代后，调节其浓度至 2.09×105个/mL，开展冷冻保存试验。Ⅰ组将STO细胞置于4 ℃下平衡，1 h后转入-20 ℃下，12 h后将其转至液氮中保存;Ⅱ组将STO细胞装入已注入250 mL 95%乙醇的降温器后，立即转至-70 ℃下，12 h后再将其转至液氮中保存;Ⅲ组将STO细胞悬液于液氮面上方10 cm处平衡20 min，然后将其缓缓转至液氮中。同时，设Ⅳ组为对照组，即STO细胞不进行冷冻处理。在液氮中保存30 d后进行解冻，以STO细胞的回收率、存活率和生长情况为研究指标，通过MTT法进行STO细胞冻存效果评价。[结果]经冷冻处理后，3种方法STO细胞解冻后回收率和存活率均存在显著差异（P<0.05）。Ⅱ组细胞解冻后细胞回收率低于Ⅰ组、高于Ⅲ组，但细胞存活率最高（84.91%）。与Ⅰ组和Ⅲ组相比，Ⅱ组STO细胞冻存方法不仅能保证冷冻效果，而且可以回收更多的细胞。Ⅱ组STO细胞复苏后生长相对缓慢，但STO细胞经培养后可恢复至冷冻前的生长状态。Ⅰ组和Ⅱ组复苏后STO细胞的增殖速度较快，且增殖率基本相同，而Ⅲ组细胞的增殖速度较慢。[结论]Ⅱ组STO细胞于-70 ℃冰箱中平衡过夜，再转移至液氮中超低温冷冻是较为理想的保存方法。

关键词 STO细胞;冷冻保存;存活率;生长

中图分类号 Q 813  文献标识码 A  文章编号 0517-6611（2021）23-0130-03

doi：10.3969/j.issn.0517-6611.2021.23.035

Effects of Different Methods on the Cryopreservation of STO Cells

TAN Ju， WU Cai-hong， WEN Chao-chao et al

（Jiangsu Agri-Animal Husbandry Vocational College，  Taizhou， Jiangsu 225300）

Abstract [Objective]To establish an optimized method for the cryopreservation of STO cells. [Method] After subculture 3 generations， the concentration of STO cells were adjusted to 2.09×105 cells per milliliter， and then the cryopreservation experiment was conducted. STO cells in I group were equalized at 4 ℃， transferred into -20 ℃ after 1 h， and stored in liquid nitrogen after 12 h. STO cells in Ⅱ group were placed in a cooler which had been injected with 250 mL 95% alcohol and then transferred into -70 ℃ immediately， and then transferred to liquid nitrogen for preservation 12 hours later. In Ⅲ group， STO cell suspension was balanced at 10 cm above the liquid nitrogen surface for 20 min， and then it was slowly transferred to liquid nitrogen.  At the same time， Ⅳ group was set as the control group， STO cells were not cryopreserved. After being stored 30 days， STO cells were thawed. The cryopreservation effect of STO cells was evaluated by using MTT method with the recovery rate， survival rate and growth rate as the research indices. [Result] After freezing treatment，  the recovery rate and survival rate of STO cells were significantly different among three different groups （P<0.05）. The recovery rate of STO cells in Ⅱ group was lower than that in I group and higher than that in Ⅲ group， but cell survival rate in Ⅱ group was the highest（84.91%）. Compared with Ⅰ group and Ⅲ group， the method of Ⅱ group could not only ensure the freezing effect， but it could also recover more cells. The growth of STO cells in Ⅱ group was relatively slow after resuscitated， but STO cells in Ⅱ group could return to the growth state before freezing. After resuscitation， the proliferation rate of STO cells in Ⅰ group and Ⅱ group was faster， and the appreciation rate was basically the same， while the proliferation rate of STO cells in Ⅲ group was slower. [Conclusion]The preservation method of group Ⅱ （STO cells were equilibrated overnight in a refrigerator at -70 ℃， and then transferred to  liquid nitrogen for ultra-low temperature freezing） was ideal for the preservation of STO cells.

Key words STO cells;Cryopreservation;Survival rate;Growth

基金项目 江苏农牧科技职业学院大学生创新创业课题（202012-806077Y）。

作者简介 谭菊（1978—），女，江苏句容人，讲师，硕士，从事兽医临床研究。

通信作者，教授，博士，从事动物生殖生物学研究。

收稿日期 2021-07-27

胚胎干细胞（ESC）因其具有自我更新能力和多向分化潜能[1]的特点而具有重要的科学价值和应用前景。……