Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells
Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells
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DOI：
10.1016/j.tice.2009.01.004
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发表时间：
2009-10-01
期刊：
影响因子：
2.6
通讯作者：
Heng, Boon Chin
中科院分区：
文献类型：
--
作者：
Heng, Boon Chin
人骨髓间充质干细胞（MSC）以前曾被报道对冻存诱导的凋亡敏感。相当大部分的MSC在冻融过程中失去活力，这是获得足够的活细胞数量以获得移植治疗最佳疗效的主要技术障碍。近年来，有报道称Rho相关激酶（ROCK）抑制剂Y-27632能提高冻存人胚胎干细胞（hESC）的冻融后活力和生理功能。因此，本研究试图研究Y-27632是否可以对冻存MSC的解冻后活力发挥类似的有益作用。在冻存培养基（10%（v/v）二甲基亚砜）以及解冻后培养基中补充浓度范围为1-100 μ M的Y-27632。补充Y-27632对解冻后即刻活力无显著影响，如通过台盼蓝拒染法评估的。然而，在冻融后24小时，将冻融的细胞悬液重新接种在新的细胞培养皿上（在解冻后培养基中具有不同浓度的Y-27632）;随后的MTT测定显示，在所检查的Y-27632浓度范围内，粘附的活细胞的比例存在显著差异，峰值在5 μ M和10 μ M之间。在Y-27632的零浓度下，活的贴壁细胞的比例为39.8 +/- 0.9%;并且在使用100 μ M Y-27632降低至36.0 +/- 0.6%之前，该值在使用5 μ M Y-27632时达到峰值48.5 +/-1.7%，在使用10 μ M Y-27632时达到峰值48.4 +/- 1.8%。此外，观察到Y-27632诱导冻融MSC的形态学变化。随着Y-27632浓度的增加，细胞显示出细胞质延伸的更广泛的分支，产生“网状”外观。这与先前关于Y-27632刺激MSC的神经元分化的报道一致。(C)2009爱思唯尔有限公司保留所有权利。
Human bone marrow-derived mesenchymal stem cells (MSC) have previously been reported to be susceptible to cryopreservation-induced apoptosis. A significant fraction of MSC lose their viability during freeze-thawing, which represent a major technical barrier in attaining adequate viable cell numbers for optimal efficacy in transplantation therapy. Recently, it was reported that a Rho-associated kinase (ROCK) inhibitor Y-27632 could enhance the post-thaw viability and physiological function of cryopreserved human embryonic stem cells (hESC). Hence, this study attempted to investigate whether Y-27632 can exert a similar beneficial effect on the post-thaw viability of cryopreserved MSC. A concentration range of 1-100 mu M Y-27632 was supplemented in both the cryopreservation medium (10% (v/v) dimethyl sulfoxide), as well as the post-thaw culture medium. The supplementation of Y-27632 had no significant effect on the immediate post-thaw viability, as assessed by trypan blue exclusion. However, 24 h after the frozen-thawed cell suspensions were re-plated on new cell culture dishes (with varying concentrations of Y-27632 within the post-thaw culture media); the MTT assay subsequently showed significant differences in the proportion of adherent viable cells over the concentration range of Y-27632 examined, with a peak at between 5 and 10 mu M. At zero concentration of Y-27632, the proportion of viable adherent cells was 39.8 +/- 0.9%; and this value peaked at 48.5 +/- 1.7% with 5 mu M Y-27632 and 48.4 +/- 1.8% with 10 mu M Y-27632, prior to decreasing to 36.0 +/- 0.6% with 100 PM Y-27632. Additionally, it was observed that Y-27632 induced morphological changes in the frozen-thawed MSC. With increasing Y-27632 concentration, the cells displayed more extensive branching of cytoplasmic extensions that gave a 'web-like' appearance. This is consistent with previous reports of Y-27632 stimulating neuronal differentiation of MSC. (C) 2009 Elsevier Ltd. All rights reserved.