Bajpai Ruchi, Lesperance Jacqueline, Kim Min, Terskikh Alexey V
Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Mol Reprod Dev. 2008 May;75(5):818-27. doi: 10.1002/mrd.20809.
Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications. However, growing and processing large quantities of undifferentiated hESCs is a challenging task. Conventionally, hESCs are passaged as clusters, which can limit their growth efficiency and use in downstream applications. This study demonstrates that hESCs can be passaged as single cells using Accutase, a formulated mixture of digestive enzymes. In contrast to trypsin treatment, Accutase treatment does not significantly affect the viability and proliferation rate of hESC dissociation into single cells. Accutase-dissociated single cells can be separated by FACS and proliferate as fully pluripotent hESCs. An Oct4-eGFP reporter construct engineered into hESCs was used to monitor the pluripotency of hESCs passaged with Accutase. Compared to collagenase-passaged hESCs, Accutase-treated cultures contained a larger proportion of undifferentiated (Oct4-positive) cells. Additionally, Accutase-passaged undifferentiated hESCs could be grown as monolayers without the need for monitoring and/or selection for quality hESC colonies.
人类胚胎干细胞（hESCs）在基于细胞的治疗和药物筛选应用方面具有巨大潜力。然而，大量培养和处理未分化的hESCs是一项具有挑战性的任务。传统上，hESCs以细胞团的形式传代，这可能会限制它们的生长效率以及在下游应用中的使用。本研究表明，使用Accutase（一种配制好的消化酶混合物），hESCs可以作为单细胞传代。与胰蛋白酶处理相比，Accutase处理对hESC解离为单细胞后的活力和增殖率没有显著影响。经Accutase解离的单细胞可以通过荧光激活细胞分选（FACS）分离，并作为完全多能的hESCs增殖。将一种Oct4-eGFP报告基因构建体导入hESCs，用于监测经Accutase传代后的hESCs的多能性。与经胶原酶传代的hESCs相比，经Accutase处理的培养物中未分化（Oct4阳性）细胞的比例更高。此外，经Accutase传代的未分化hESCs可以作为单层生长，无需监测和/或选择高质量的hESC集落。
