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Surface-Based Cryopreservation Strategies for Human Embryonic Stem Cells: A Comparative Study

机译：基于表面的人类胚胎干细胞冷冻保存策略：一项比较研究

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Human embryonic stem cells (hESC) hold tremendous potential in the emerging fields of gene and cell therapy as well as in basic scientific research. One of the major challenges regarding their application is the development of efficient cryopreservation protocols for hESC since current methods present poor recovery rates and/or technical difficulties which impair the development of effective processes that can handle bulk quantities of pluripotent cells. The main focus of this work was to compare different strategies for the cryopreservation of adherent hESC colonies. Slow-rate freezing protocols using intact hESC colonies was evaluated and compared with a surface-based vitrification approach. Entrapment within ultra-high viscous alginate was investigated as the main strategy to avoid the commonly observed loss of viability and colony fragmentation during slow-rate freezing. Our results indicate that entrapment beneath a layer of ultra-high viscous alginate does not provide further protection to hESC cryopreserved through slow-rate freezing, irrespectively of the cryomedium used. Vitrification of adherent hESC colonies on culture dishes yielded significantly higher recovery rates when compared to the slow-rate freezing approaches investigated. The pluripotency of hESC was not changed after a vitrification/thawing cycle and during further propagation in culture. In conclusion, from the cryopreservation methods investigated in this study, surface-based vitrification of hESC has proven to be the most efficient for the cryopreservation of intact hESC colonies, reducing the time required to amplify frozen stocks thus supporting the widespread use of these cells in research and clinical applications.
机译：人类胚胎干细胞（hESC）在新兴的基因和细胞治疗领域以及基础科学研究中具有巨大潜力。关于它们的应用的主要挑战之一是开发用于hESC的有效的低温保存方案，因为当前的方法存在回收率低和/或技术困难，这损害了可以处理大量多能细胞的有效方法的开发。这项工作的主要重点是比较粘附hESC菌落的冷冻保存的不同策略。使用完整的hESC菌落的慢速冷冻方案进行了评估，并与基于表面的玻璃化方法进行了比较。研究了超高粘度藻酸盐内的截留是避免通常观察到的慢速冷冻过程中生存力丧失和菌落破碎的主要策略。我们的结果表明，无论使用何种冷冻液，在超高粘性藻酸盐层下的截留都不能进一步保护通过慢速冷冻冷冻保存的hESC。与研究的慢速冷冻方法相比，附着在培养皿上的hESC菌落的玻璃化可显着提高回收率。在玻璃化/解冻循环之后以及在培养物中进一步繁殖期间，hESC的多能性没有改变。总之，从本研究中研究的冷冻保存方法中，已证明基于hESC的表面玻璃化对完整的hESC菌落进行冷冻保存是最有效的，从而减少了扩增冷冻原种所需的时间，从而支持了这些细胞在哺乳动物中的广泛使用。研究和临床应用。

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