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Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

机译：人脐带间充质干细胞分离培养方法的优化

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Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16–20 h is an effective and simple modified enzyme digestion method.Electronic supplementary materialThe online version of this article (doi:10.1007/s10616-012-9528-0) contains supplementary material, which is available to authorized users.
机译：人脐带间充质干细胞（hUCMSC）被认为是骨髓MSC的理想替代品。然而，迄今为止，还没有用于hUCMSC分离和培养的简便有效的方法。本研究旨在探索改良的hUCMSC体外酶切方法。在hUCMSCs上使用常规酶消化，修饰的酶消化和组织外植体，比较它们的分离和培养效率，观察原代细胞生长和细胞亚培养。结果表明，与两种酶消化方法相比，使用组织外植体方法培养的细胞具有更长的培养周期（P <0.01）和每厘米脐带的原代细胞产量较低（P <0.01）。组织移植法与常规酶消化法相比，亚培养粘附和细胞倍增所需的时间显着减少（P <0.05）;但是，组织外植体法和改良酶消化法之间没有显着差异（P> 0.05）。比较两种酶消化方法，改良方法比传统方法产生的细胞更多（P <0.01），改良方法比传统方法产生的原代细胞粘附时间显着减少（P <0.05）。用改良酶消化法培养的第三代hUCMSCs的细胞周期分析表明，大多数细胞处于静止状态。免疫荧光染色显示这些细胞表达了MSC标记CD44和CD90。冯·科萨（Von Kossa）和油红O染色检测表明，在体外诱导培养基中它们可以分化为成骨细胞和脂肪细胞。这项研究表明，hUCMSC的分离和培养在37°C下使用0.2％胶原酶II进行16-20h的消化是一种有效且简单的改良酶消化方法。电子补充材料本文的在线版本（doi：10.1007 / s10616-012） -9528-0）包含补充材料，授权用户可以使用。

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