An in vitro Mechanical Damage Model of Isolated Myofibers in a Floating Culture Condition.

Muscle stem cells (satellite cells), located on the surface of myofibers, are rapidly activated from a quiescent state following skeletal muscle injury. Although satellite cell activation is an initial step in muscle regeneration, the stimulation of satellite cell activation by muscle injury remains to be elucidated. We recently established an in vitro mechanical damage model of myofibers, to analyze quiescent and activated satellite cells associated with myofibers isolated from the extensor digitorum longus muscle in mice. Here, we described a protocol for the mechanical damage of myofibers and co-culture of intact healthy myofibers with damaged myofibers in a floating condition. This in vitro myofiber damage model allowed us to investigate the mechanism of satellite cell activation without contamination by interstitial cells, such as blood vessel cells and fibroblasts, as well as understand how damaged myofiber-derived factors (DMDFs) activate satellite cells.