Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique
Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique
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DOI：
10.1038/nprot.2008.142
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Huard, Johnny
中科院分区：
文献类型：
--
作者：
Gharaibeh, Burhan;Lu, Aiping;Huard, Johnny
该方案详细介绍了一种被称为改进的预板技术的程序，该技术目前在我们实验室用于分离肌肉细胞，其基础是选择性地黏附于胶原蛋白涂层的组织培养板。通过将这一技术应用于小鼠骨骼肌，我们已经能够在该过程的早期阶段分离出快速黏附细胞(RAC)部分，而从该过程的后期阶段获得含有肌源性干细胞的缓慢黏附细胞(SAC)部分。该方案概述了从小鼠骨骼肌中分离RAC和SAC种群所需的方法和材料。这一过程包括用胶原酶XI、消解酶和胰酶机械和酶消化骨骼肌组织，然后将生成的肌浆涂在I型胶原涂层的烧瓶上，细胞以不同的速度粘连。整个预制板技术需要5d来获得最终的预制板SAC密度。通常还需要两到三天的额外时间才能适当地建立这一群体。我们还详细介绍了其他方法，旨在通过继续对SAC群体进行修改的预平板过程来进一步丰富所得到的细胞群体。这个过程被称为重复，需要更多的时间。
This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.