人肺腺癌细胞A549低温保存优化研究

人肺腺癌A549细胞广泛用于体外肺癌发病基因筛查、药物筛选等临床研究，低温保存是保持样本库中的A549细胞高质量的有效方法。本文针对A549细胞低温保存方法，通过使用非接触置核慢速降温程序减小降温过程中溶液的过冷度，并在此基础上优化保护剂乙二醇（ethylene glycol，EG）、二甲基亚砜（dimethyl sulfoxide，Me2SO）以及海藻糖的最佳浓度，通过检测复苏后肺腺癌细胞的回收率、细胞凋亡情况以及凋亡相关基因表达，对保存效果进行评估。实验结果表明，非接触置核慢速降温程序相比传统慢速降温程序可有效降低低温保护剂过冷度，并提高了细胞回收率。优化后最佳低温保护剂组合为7.5%（体积分数）Me2SO+2.5%（体积分数）EG+200 mmol 海藻糖，使用该保护剂低温保存复苏后的细胞回收率（91.59±1.51%）高于使用传统低温保护剂10%（体积分数）Me2SO的细胞回收率（86.72 ±1.86%），而且正常细胞比例高，可以很好地抑制细胞凋亡。

Optimization of the cryopreservation for human lung adenocarcinomacell lines A549

A549 cell is a good model for screening apoptosis-inducing drugs for lung adenocarcinoma in vitro research. It is of great significance to maintain high-activity and high-quality A549 cells in the biobank. In the paper, a non-contact ice-seeding slow cooling program were adopted to reduce the subcooling of the cryoprotectant(CPA) during the cooling process. The concentration of EG, Me₂SO and trehalose were optimized for the cryopreservation of A549 cells. The results show that the non-contact ice-seeding cooling program can effectively reduce the subcooling of the cryoprotectant compared to the traditional slow cooling program; the optimized cryoprotectant combination is 7.5%(V/V)Me₂SO+2.5%(V/V)EG+200 mmol trehalose. After cryopreservation and rewarming, the recovery rate of the cells using this CPA is higher than the traditional cryoprotectant of 10%(V/V) Me₂SO (91.59±1.51% vs 86.72±1.86%). The proportion of normal cells is high, which indicates that the combined cryoprotectant can effectively inhibit apoptosis after cryopreservation.