Isolation, growth and differentiation of adult rabbit skeletal myoblasts in vitro

A simple and reliable method is described for the growth and differentiation of myoblasts isolated from adult New Zealand White rabbit fast-twitch (tibialis anterior) and slow-twitch (soleus) skeletal muscle. Cells were dissociated mechanically, and expanded in DMEM supplemented with 20% horse serum. The myoblasts were differentiated by switching to DMEM supplemented with 10% horse serum when the myoblasts were 80–90% confluent. The myoblasts fused into multinucleated myotubes that spontaneously contracted. At the light microscopic level, the myotubes exhibited a striated pattern as revealed by positive immunostaining for sarcomeric proteins such as -actinin, myosin and titin.Electron microscopy demonstrated well ordered sarcomeres with localized mitochondria and sarcoplasmic reticulum. The only notable difference between the fiber types was in the initial response to isolation wherein the myoblast yield was four-fold greater from the soleus than the tibialis anterior, in agreement with satellite cell abundance in predominately slow-twitch vs fast-twitch muscles. These techniques repeatedly (n = 12) produced a population of healthy myoblasts isolated from either fast- or slow-twitch skeletal muscle which can be utilized for studies of skeletal muscle differentiation, assembly and signaling.