Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells

Department of Biological Structure, School of Medicine, University of Washington, Seattle, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2005; 290:281-304. DOI: 10.1007/978-1-62703-128-8_28
Source: PubMed


Myofibers are the functional contractile units of skeletal muscle. Mononuclear satellite cells located between the basal lamina and the plasmalemma of the myofiber are the primary source of myogenic precursor cells in postnatal muscle. This chapter describes protocols used in our laboratory for isolation, culturing, and immunostaining of single myofibers from mouse skeletal muscle. The isolated myofibers are intact and retain their associated satellite cells underneath the basal lamina. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. Myofibers are cultured in dishes coated with Vitrogen collagen, and satellite cells remain associated with the myofibers undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL). Different from the FDB myofibers, the longer EDL myofibers tend to tangle and break when cultured together; therefore, EDL myofibers are cultured individually. These myofibers are cultured in dishes coated with Matrigel. The satellite cells initially remain associated with the myofiber and later migrate away to its vicinity, resulting in extensive cell proliferation and differentiation. These protocols allow studies on the interplay between the myofiber and its associated satellite cells.

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    • "In the first strategy, satellite cells were allowed to branch out of muscle fibers and attach to the dish bottom. In the second strategy, satellite cells were isolated as a " pure " culture by enzymatic dissociation of muscle fibers [28] [36] [37]. For the first strategy, muscle fibers were isolated from soleus and flexor digitorum brevis muscles by incubation in collagenase and subsequent trituration as described above and incubated until the satellite cells appeared in the dishes. "
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    • "Isolation of single mature skeletal muscle fibers Single fibers were isolated from the FDB muscles of mice [28]. "
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    • "Satellite cells were isolated as described elsewhere [26]. Muscles were digested in 0.1% collagenase I (Sigma, Germany) for 90 min at 37 • C. "
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