ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells
ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells
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DOI：
10.1093/humrep/den404
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发表时间：
2009-03-01
影响因子：
6.1
通讯作者：
Rancourt, Derrick E.
中科院分区：
文献类型：
--
作者：
Li, Xiangyun;Krawetz, Roman;Rancourt, Derrick E.
在无血清和无饲养层培养系统中有效的缓慢冷冻方案对于人胚胎干细胞（hES）的临床应用至关重要。然而，当使用常规的缓慢冷冻方案时，hES细胞通常必须作为团块冷冻保存，导致冻融期间存活率较低，并限制了解冻后的回收和生长效率，以及限制了需要单细胞悬浮液的下游应用。我们描述了一种新的方法，以提高冻融存活和单个hES细胞在无血清和无饲养层培养条件下的增殖率。在37 ℃解冻后，将细胞在补充有10 μ M Rho相关激酶抑制剂Y-27632的mTeSR培养基中培养1天。与对照组相比，使用Y-27632和Accutase显著增加解冻后单个hES细胞的存活率（P < 0.01）。此外，通过用EGTA处理hES细胞聚集体以破坏细胞-细胞相互作用，我们表明Y-27632处理不直接影响hES细胞凋亡。即使在Y-27632的存在下，缺乏细胞-细胞相互作用的hES细胞也经历凋亡。Y-27632处理的冻融hES细胞保持典型的形态学、稳定的核型，多能性标记的表达和在长期培养后分化成所有三个胚层的衍生物的潜力。因此我们相信该方法对于当前和未来的靶向治疗终点的hES细胞应用将是理想的。
Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently, however, hES cells must be cryopreserved as clumps when using conventional slow freezing protocols, leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing, as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions.hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C, cells were cultured in mTeSR medium supplemented with 10 mu M of Rho-associated kinase inhibitor Y-27632 for 1 day.The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P < 0.01). Furthermore, by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction, we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632, hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture.The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point.