A Modified Pre-plating Method for High-Yield and High-Purity Muscle Stem Cell Isolation From Human/Mouse Skeletal Muscle Tissues
A Modified Pre-plating Method for High-Yield and High-Purity Muscle Stem Cell Isolation From Human/Mouse Skeletal Muscle Tissues
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DOI：
10.3389/fcell.2020.00793
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发表时间：
2020-08-13
影响因子：
5.5
通讯作者：
Ono, Yusuke
中科院分区：
文献类型：
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作者：
Yoshioka, Kiyoshi;Kitajima, Yasuo;Ono, Yusuke
骨骼肌干细胞的原代培养是研究肌肉再生动力学和稳态不可或缺的。在这里，我们描述了用于分离培养物中的MuSC的改进的预接种方法，其具有大大提高的纯度、产率和操作时间。该方案基于MuSC的独特粘附特性。我们将程序时间缩短至2.5天，以通过新采用的重新铺板步骤获得高度纯化的MuSC，该步骤重复孵育和细胞悬浮。重新铺板步骤有效地将剩余的成纤维细胞而不是MuSC捕获在胶原包被的培养皿上。此外，我们证实了MuSCs可以从少量的人/小鼠肌肉组织中分离出来，使我们能够用有限的标本进行实验。因此，我们的方法可以用适用于大多数设施的基本实验室设备进行，而无需复杂的MuSC处理技术。
Primary culture of skeletal muscle stem cells (MuSCs) is indispensable to study the dynamics of muscle regeneration and homeostasis. Here we describe the modified pre-plating method for isolating MuSCs in culture with greatly improved purity, yield, and procedure time. The protocol is based on the distinct adhesion characteristics of MuSCs. We reduced the procedure time to 2.5 days to obtain highly purified MuSCs through a newly employed re-plating step, which repeats incubation and cell-suspension. The re-plating step efficiently traps remaining fibroblastic cells, but not MuSCs, on a collagen-coated dish. Additionally, we confirmed that MuSCs can be isolated from small amounts of human/mouse muscle tissues, enabling us to perform experiments with amount-limited specimens. Thus, our method can be performed with basic laboratory equipment suitable for most facilities and without sophisticated MuSC handling techniques.