Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden.
Hum Reprod. 2010 May;25(5):1271-9. doi: 10.1093/humrep/deq040. Epub 2010 Mar 5.
Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) bear a great potential in regenerative medicine. In addition to optimized clinical grade culture conditions, efficient clinical grade cryopreservation methods for these cells are needed. Obtaining good survival after thawing has been problematic.
We used a novel, chemically defined effective xeno-free cryopreservation system for cryostorage and banking of hESCs and iPSCs. The earlier established slow freezing protocols have, even after recent improvements, resulted in low viability and thawed cells had a high tendency to differentiate. The medium is a completely serum and animal substance free product containing dimethylsulfoxide, anhydrous dextrose and a polymer as cryoprotectants. The cells were directly frozen at -70 degrees C, without a programmed freezer.
The number of frozen colonies versus the number of surviving colonies differed significantly for both HS293 (chi(2) = 9.616 with one degree of freedom and two-tailed P = 0.0019) and HS306 (chi(2) = 8.801 with one degree of freedom and two-tailed P = 0.0030). After thawing, the cells had a high viability (90-96%) without any impact on proliferation and differentiation, compared with the standard freezing procedure where viability was much lower (49%). The frozen-thawed hESCs and iPSCs had normal karyotype and maintained properties of pluripotent cells with corresponding morphological characteristics, and expressed pluripotency markers after 10 passages in culture. They formed teratomas containing tissue components of the three germ layers.
The defined freezing-thawing system described here offers an excellent simple option for banking of hESCs and iPSCs.
人类胚胎干细胞（hESCs）和诱导多能干细胞（iPSCs）在再生医学方面具有巨大的潜力。除了优化临床级别的培养条件外，还需要高效的临床级别的这些细胞的冷冻保存方法。解冻后获得良好的存活率一直是个问题。
我们使用了一种新颖的、化学定义的、有效的无动物源的冷冻保存系统来冷冻保存和储存 hESCs 和 iPSCs。早期建立的慢速冷冻方案，即使经过最近的改进，也导致存活率低，解冻后的细胞有很高的分化倾向。该培养基是一种完全不含血清和动物物质的产品，含有二甲亚砜、无水葡萄糖和一种聚合物作为冷冻保护剂。细胞直接在-70°C 下冷冻，无需程序冷冻器。
对于 HS293（chi(2) = 9.616，自由度为 1，双侧 P = 0.0019）和 HS306（chi(2) = 8.801，自由度为 1，双侧 P = 0.0030），冷冻的菌落数与存活的菌落数有显著差异。与标准冷冻程序相比，解冻后的细胞存活率很高（90-96%），对增殖和分化没有任何影响，而标准冷冻程序的存活率要低得多（49%）。冷冻-解冻的 hESCs 和 iPSCs 具有正常的核型，并保持多能细胞的特性，具有相应的形态特征，并在培养 10 代后表达多能性标记。它们形成含有三个胚层组织成分的畸胎瘤。
本文描述的定义明确的冷冻-解冻系统为 hESCs 和 iPSCs 的储存提供了一个极好的简单选择。
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