Bulk vitrification of human embryonic stem cells
Bulk vitrification of human embryonic stem cells
复制标题
人类胚胎干细胞的批量玻璃化冷冻。
DOI：
10.1093/humrep/dem386
复制
发表时间：
2008-02-01
影响因子：
6.1
通讯作者：
Zhuang, Guanglun
中科院分区：
文献类型：
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作者：
Li, Tao;Zhou, Canquan;Zhuang, Guanglun
背景
传统的玻璃化冷冻方法无法满足冷冻保存大量增殖旺盛的人胚胎干细胞（HES）所需的培养和繁殖效率的提高。在这项研究中，我们描述了一种新发明的玻璃化载体冷冻保存大量的羟乙基淀粉细胞，并评估这种散装玻璃化（BV）方法是否是有效的流行的开放式吸管（OPS）玻璃化方法。
方法
传代后收获HES细胞团，转移至细胞滤器中;仅直径大于70 μ m的细胞团才纳入研究，并随机选择BV法、OPS玻璃化法或慢速冷冻法进行冻存。解冻后分析HES细胞存活、生长和多能性。
结果
细胞过滤器玻璃化法一次可冻存136 ± 23.4个细胞团，是OPS法（4 ± 1.5个）的30倍。大体积玻璃化冷冻的HES细胞解冻后的存活率高达94.3%，与OPS法相当。所有存活的细胞团块产生HES细胞集落。在皮下注射解冻后的大量玻璃化的HES细胞团块后，在严重联合免疫缺陷小鼠中形成了包含所有三个原始胚层的畸胎瘤，证实了多能性。
结论
这种新的BV法可以一次性冻存大量的HES细胞团，不仅可以满足日常培养过程中HES细胞的常规冻存，而且可以保证研究人员随时有大量高效冻存的HES细胞用于预定的研究。
BACKGROUND
The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method.
METHODS
HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing.
RESULTS
Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency.
CONCLUSIONS
This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.