Sheep Myoblast Isolation and Differentiation

According to previous studies, sheep myoblasts were isolated by a two-step enzymatic method using muscle from newborn 5-day-old lambs [7 (link)]. Briefly, leg muscles were cut into small pieces and washed three times with DPBS, digested with 0.1% type I collagenase (Sigma-Aldrich, Saint Louis, MO, USA) for 1 h, and then digested with 0.25% trypsin (Gibco, Grand Island, NY, USA) for 30 min. The tubes were shaken every 10 min and filtered through a 200-mesh sieve. The cells were cultured in a growth medium consisting of DMEM-F12 (Gibco, Grand Island, NY, USA) supplemented with 20% FBS and 10% heat-inactivated horse serum (Gibco, Grand Island, NY, USA). Two hours later, the cell supernatant was transferred to a new flask and the cells began to adhere after 48 h. Myoblasts within four generations were used for subsequent studies. Differentiation of the myoblasts was carried out in a medium containing 2% horse serum in DMEM-F12. The differentiation was observed at 0, 72, and 120 h after differentiation.