Sincennes Marie Claude, Wang Yu Xin, Rudnicki Michael A
Sprott Center For Stem Cell Research, Ottawa Hospital Research Institute, Regenerative Medicine Program, 501 Smyth, Box 511, Ottawa, ON, Canada, K1H 8L6.
Faculty of Medicine, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.
Methods Mol Biol. 2017;1556:41-50. doi: 10.1007/978-1-4939-6771-1_3.
Primary myoblasts can be isolated from mouse muscle cell extracts and cultured in vitro. Muscle cells are usually dissociated manually by mincing with razor blades or scissors in a collagenase/dispase solution. Primary myoblasts are then gradually enriched by pre-plating on collagen-coated plates, based on the observation that mouse fibroblasts attach quickly to collagen-coated plates, and are less adherent. Here, we describe an automated muscle dissociation protocol. We also propose an alternative to pre-plating using magnetic bead separation of primary myoblasts, which improve myoblast purity by minimizing fibroblast contamination.
原代成肌细胞可从小鼠肌肉细胞提取物中分离出来并进行体外培养。肌肉细胞通常在胶原酶/分散酶溶液中用剃须刀片或剪刀切碎进行手动解离。然后，基于小鼠成纤维细胞能快速附着在胶原包被的培养板上且贴壁性较差这一观察结果，通过在胶原包被的培养板上预铺板来逐步富集原代成肌细胞。在此，我们描述一种自动肌肉解离方案。我们还提出一种使用磁珠分离原代成肌细胞替代预铺板的方法，该方法通过尽量减少成纤维细胞污染来提高成肌细胞纯度。
