Optimization of a protocol for the high-yield isolation of active muscle stem cells from bovine skeletal muscle tissue.

Beef is primarily made up of skeletal muscle tissue. Therefore, the cultivation of bovine muscle stem cells (MSCs) to provide a consistent supply of muscle cells would enhance the sustainability of the cultured beef industry. Here, we report a high-yield, simple, economic, and convenient protocol for the isolation of active MSCs from bovine skeletal muscle tissue. We optimized the enzymatic tissue dissociation protocol and the composition of the medium used for differential plating (DP) to enhance the purity of active MSCs isolated from primary cells derived from the tissue. In addition, the optimal source of bovine muscle tissue for the isolation of active MSCs was determined. The yield of active MSCs was maximized by incubating round area-derived skeletal muscle tissue for 30 min in 0.2% (w/v) collagenase type II in high-glucose DMEM (HG-DMEM), followed by 1% (w/v) pronase in HG-DMEM for 5 min, and conducting DP of the enzymatically dissociated skeletal muscle tissue-derived primary cells in HG-DMEM supplemented with 10% (v/v) FBS and 5 ng/mL bFGF. In conclusion, we established a simple, convenient, and inexpensive protocol for the high-yield isolation of active MSCs from bovine skeletal muscle tissue. This protocol could overcome the technical challenges that hamper the large-scale production of bovine muscle cells, thereby enabling the commercialization of cultured beef.