Background

Muscle stem cells called satellite cells reside between the plasmalemma of myofibers and the basal lamina, and maintain a quiescent state in healthy muscles (Mauro, 1961). Satellite cells play important roles in postnatal muscle growth, hypertrophy, and regeneration, by providing new myonuclei of myofibers in adults. Following muscle injury, satellite cells are rapidly activated from their quiescent state to become myoblasts. Then, they proliferate to give rise to progeny, and fuse with one another and/or with existing damaged myofibers to regenerate muscles. Although the activation of satellite cells is an initial step in muscle regeneration, its mechanism remains to be elucidated.

Extracts from crushed muscle tissues were observed to stimulate the activation and proliferation of cultured myogenic cells (Vandenburgh et al., 1984; Kardami et al., 1985; Bischoff, 1986; Mezzogiorno et al., 1993; Haugk et al., 1995), indicating that muscle tissues contain an activator of satellite cells. Consequently, cytokines and growth factors, including hepatocyte growth factor, have been identified as activators of satellite cells (Tatsumi et al., 1998; Li, 2003; Zeng et al., 2010). Muscle tissues are composed of myofibers as well as various types of other cells, such as interstitial mesenchymal cells and immune cells (e.g., blood vessel cells, macrophages, and fibroblasts) (Evano and Tajbakhsh, 2018). Thus, such non-muscle cell-derived factors may directly and indirectly influence satellite cell dynamics and contribute to muscle regeneration.

The individual myofiber isolation and culture technique is a useful tool to analyze satellite cell fate dynamics, ranging from quiescence to activation, proliferation, differentiation, and self-renewal in their niche of myofibers, without contamination by interstitial cells (Zammit et al., 2004; Ono et al., 2011). By applying this culture system, we recently established an in vitro mechanical damage model of isolated myofibers in a floating culture condition. This model can be applied to the co-culture of intact myofibers harboring satellite cells with mechanically damaged myofibers, allowing us to examine how damaged myofiber-derived factors (DMDFs) influence satellite cell dynamics (Tsuchiya et al., 2020), and better understand the mechanism of the initial step of muscle regeneration. Here, we describe a detailed protocol for the isolation and mechanical damage of individual myofibers isolated from the extensor digitorum longus (EDL) muscle in mice, as well as floating co-culture of intact myofibers and damaged myofibers. Our in vitro mechanical myofiber damage model is useful for developing efficient regeneration therapies for muscle wasting diseases, such as muscular dystrophies and age-related sarcopenia, as well as for establishing a strategy for rapid recovery from severe muscle injury in athletes.