Ice recrystallization inhibitors enable efficient cryopreservation of induced pluripotent stem cells: A functional and transcriptomic analysis

The successful use of human induced pluripotent stem cells (iPSCs) for research or clinical applications requires the development of robust, efficient, and reproducible cryopreservation protocols. After cryopreservation, the survival rate of iPSCs is suboptimal and cell line dependent. We assessed the use of ice recrystallization inhibitors (IRIs) for cryopreservation of human iPSCs. A toxicity screening study was performed to assess specific small-molecule carbohydrate-based IRI and concentrations for further evaluation. Then, a cryopreservation study compared the cryoprotective efficiency of 15 mM IRIs in 5 % or 10 % DMSO-containing solutions and with CryoStor® CS10. Three iPSC lines were cryopreserved as single-cell suspensions in the cryopreservation solutions and post-thaw characteristics, including pluripotency and differential gene expression, were assessed. We demonstrate the fitness-for-purpose of 15 mM IRI in 5 % DMSO as an efficient cryoprotective solution for iPSCs in terms of post-thaw recovery, viability, pluripotency, and transcriptomic changes. Given that this dataset is the first report where mRNA sequencing has been used to identify expression changes resulting from iPSCs cryopreservation, it has the potential to be used for molecular mechanism analysis relating to cryopreservation. IRIs can reduce DMSO concentrations, thereby improving the utility, effectiveness, and efficiency of cryopreservation.将人类诱导多能干细胞（induced pluripotent stem cells, iPSCs）应用于研究或临床场景并取得成功，需建立稳健、高效且可重复的冷冻保存方案。经冷冻保存后，iPSCs的存活率仍未达最优水平，且呈细胞系依赖性。本研究评估了冰重结晶抑制剂（ice recrystallization inhibitors, IRIs）在人类iPSCs冷冻保存中的应用效果：首先开展毒性筛选实验，以筛选适用于后续评估的特定小分子碳水化合物类冰重结晶抑制剂及其使用浓度；随后开展冷冻保存实验，对比15 mM冰重结晶抑制剂在含5%或10%二甲基亚砜（dimethyl sulfoxide, DMSO）的溶液中，以及在CryoStor® CS10溶液中的冷冻保护效率。本研究将3株iPSCs以单细胞悬液的形式置于上述冷冻保存溶液中进行冷冻保存，并对解冻后的细胞特性进行评估，包括多能性及差异基因表达情况。本研究证实，在含5%二甲基亚砜的溶液中添加15 mM冰重结晶抑制剂，可作为一款符合使用要求的高效iPSCs冷冻保护液，其在解冻后细胞回收率、细胞活力、多能性及转录组变化等各项指标上均表现优异。由于本数据集是首个利用mRNA测序（messenger RNA sequencing）技术鉴定iPSCs冷冻保存所致基因表达变化的相关报道，其有望用于冷冻保存相关分子机制的分析研究。冰重结晶抑制剂可降低二甲基亚砜的使用浓度，从而提升冷冻保存的实用性、有效性与效率。