Article

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

ABSTRACT

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.

    • "Given that the muscle tissue is directly minced, the result is a myoblast culture containing of a variety of cell types, such as fibroblasts and adipocytes. Alternatively, purer myoblast cultures can be achieved through the isolation of live myofibers (Keire, Shearer, Shefer, & Yablonka-Reuveni, 2013), to which satellite cells are still attached. In culture, the satellite cells can be allowed to migrate out from the fiber, or alternatively, the myofibers can be grown in floating conditions, allowing satellite cells to activate and proliferate on the surface of the myofibers in a more natural environment. "
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    • "First, in ex vivo culturing experiments, we did not observe significant changes in domain size or dissipation of the mito-Dendra2 signal over the course of 72 hr (Figures S4A and S4B). Thus, mitochondrial domains are stable under standard culturing conditions that maintain myofibers in a healthy and functional state (Casas et al., 2010;Eisner et al., 2014;Keire et al., 2013). Second, we observed a significant increase in the frequency of mitochondrial domains in aged animals, suggesting that domains are stably accumulating over time (Figures S4C and S4D). "
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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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