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

Abstract

Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing, and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, 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) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.

Fig. 1

A schematic A and EM micrograph B of satellite cell location. The myofiber basement and plasma membranes have been routinely detected by immunostaining with antibodies against laminin and dystrophin, respectively. In panel A, myofiber nuclei depicted at the myofiber periphery represent the state of healthy adult myofibers (while immature myofibers present in regenerating muscles display centralized myofiber nuclei; not shown). In panel B, black arrows depict the basal lamina, white arrows depict apposing satellite cell and myofiber membranes; note the sarcomeric organization within the myofiber.

Fig. 2

Parallel 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 in solution (now known as PureCol). Cultures were maintained for 4 days in basal medium containing fibroblast growth factor 2 (FGF2, 2 ng/mL) and fixed with methanol as described in Subheading 3.3.1. A & B Phase and DAPI stained images (both myofiber nuclei and satellite cell nuclei are labeled with DAPI). C & D Myofiber culture reacted by double immunofluorescence with a monoclonal antibody against myogenin (identifies the nuclei of differentiated myogenic cells) and a polyclonal antibody immunostaining against ERK1/ERK2 mitogen activated protein kinases (MAPK) (identifies the cytoplasm of all fiber-associated cells). Reactivity with the monoclonal and polyclonal antibodies was traced with fluorescein- and rhodamine-labeled secondary antibodies, respectively. 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 400x magnification. Additional details regarding the source of the antibodies and the rationale of using these antibodies are provided in our previous publications (22,35,36,46);.

Fig. 3

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 in Subheading 3.3.2. Satellite cells begin to emigrate from the myofiber within the first day in culture and continue to emigrate during subsequent days. Progeny of satellite cells that have emigrated from the myofibers proliferate, differentiate and fuse into myotubes, establishing a dense myogenic culture. A Satellite cells remained attached to the muscle fiber during the first hours after culturing. B Nineteen hours after culturing, 2–3 cells detached from the fiber but remained in close proximity to the fiber. C Four days following culturing more cells are seen in the vicinity of the myofibers (at least 4 cells are visible). D By day 7, progeny of satellite cells that emigrated from the myofiber have established a culture containing mostly proliferating myoblasts and some myotubes. Micrographs in panels A-C were taken with a 400x magnification to show details of the few cells that emigrated from the myofiber, while the micrograph in panel D was taken with a 100x magnification to show the establishment of a dense myogenic culture. See our published study for additional details about growth of satellite cell progeny in long-term EDL myofiber cultures (22);.

Fig. 4

Dissection of FDB muscle from the rear foot of adult mouse. A Rear foot before dissection. B Cutting of “T” toward the ankle, left to right); arrowheads identify the circumferential cut at the ankle and arrow shows the direction of cutting. C Peeling the skin back from the ankle exposing the muscles and tendons. D Digit tendons of the FDB exposed on the sole of the foot. E Cutting the connective tissue under the FDB toward the heal of the foot. F Freeing the FDB from the underlying connective tissue. G Cutting the FDB at the heal origin; arrow indicates direction of cutting. (H) Preparing the release of the FDB from its tendon insertion points at the digits.

Fig. 5

Dissection of EDL muscle from the hindlimb of adult mouse. A Anterior lower hindlimb with skin removed. B Facia covering the anterior lower hindlimb muscles is removed to allow access to tendons. C The four foot tendon insertion points of the EDL are isolated and cut. D The common tendon of the EDL is carefully exposed and isolated at the ankle. E Once isolated and foot insertions are cut, the EDL tendons are pulled proximally up from the foot; arrows indicate the direction of pulling. The tendons should easily slide underneath the connective tissue sheath at the ankle up from the foot. If the tendons do not easily slide out, then reexamine the foot tendons to ensure that they have been cut. F Origin of the EDL is exposed then cut at the lateral surface of the tibia condyle head. (G Grasping only the EDL tendon (do not grasp the muscle as it can easily be damaged), carefully pull distally toward the toes to remove the EDL muscle; arrow indicates the direction of pulling. H) The EDL should slide underneath the TA muscle and should pull out easily. It is important to pull gently and there should be little resistance; if the muscle does not slide out easily, one or both tendons at the muscle origin may still be attached to the bone. In this case identify the attached tendon and cut it.