Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells

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.

Fig 1

Phase and immunofluorescent micrographs of an isolated FDB myofiber with associated satellite cells undergoing myogenesis. Myofibers were isolated from a 3-month-old mouse and cultured in 35-mm tissue culture dishes coated with isotonic Vitrogen collagen. Cultures were maintained for 4 days in basal medium containing fibroblast growth factor 2 (FGF2, 2 ng/ml) and fixed with methanol as described under subheading 3.3.1.1. The culture shown in this figure was reacted via double immunofluorescence with a monoclonal antibody against myogenin which stains the nuclei of myogenic cells that have entered the differentiated step of myogenesis (panel C) and a polyclonal antibody against ERK1/ERK2 mitogen activated protein kinases (MAPK), which stains the cytoplasm of all fiber-associated cells (panel D). Reactivity with the monoclonal and polyclonal antibodies was traced with a fluorescein- and rhodamine- labeled secondary antibody, respectively. Parallel phase image (panel A) and DAPI staining image (panel D; both myofiber nuclei and satellite cell nuclei are stained) are shown as well. Arrows in parallel panels point to the location of the same cell. Additional immunopositive cells present on the myofiber are not shown, as not all positive nuclei or cells on the fibers are in the same focal plane. All micrographs were taken with a 40x objective. Additional details regarding the source of the antibodies and the rationale of using these antibodies are provided in our previous publications (10,15).

Fig. 2

Phase micrographs of EDL myofibers depicting the temporal development of myogenic cultures from cells emanating from individual myofibers. Myofibers were isolated from 3 month- old mice and cultured individually in 24-well multiwell tissue culture dishes coated with Matrigel. Cultures were maintained in serum-rich/mitogen-rich growth medium and fixed with paraformaldehyde, as described under subheading 3.3.1.2. Satellite cells begin to emigrate from the myofiber within the first day in culture and continue to emigrate during subsequent days. Satellite cells that have emigrated from the myofibers proliferate, differentiate and fuse into myotubes, establishing a dense myogenic culture. Satellite cells remained attached to the muscle fiber during the first hours after culturing (panel A). Nineteen hours after culturing, 2-3 cells detached from the fiber but remained in close proximity to the fiber (panel B). Four days following culturing more cells are seen in the vicinity of the myofibers (only 4 cells shown in panel C). By day 7 progeny of satellite cells that emigrated from the myofiber have established a culture containing mostly proliferating myoblasts and some myotubes (panel D). Micrographs in panels A-C were taken with a 40x objective to show details of the few cells that emigrated from the myofiber, while the micrograph in panel D was taken with a 10x objective to show the establishment of a dense myogenic culture