Isolation and Culture of Human Muscle Progenitor Cells

Human muscle biopsies from the M. rectus abdominis were collected upon ethical approval and with informed consent of 6 hospitalized patients undergoing abdominal surgery under general anesthesia. The patients were selected according to strict inclusion and exclusion criteria, assuring the optimal quality of the muscle biopsy (e.g., no muscular dystrophy, hormonal therapy, and chronic infectious diseases). The samples were processed according to established protocols [29 (link)]. Briefly, each muscle biopsy was first minced and digested with collagenase type I 0.2% (w/v) (Sigma) and dispase 0.4% (w/v) (Gibco). The enzymatic reaction was terminated with medium containing 10% FBS. Individual fibers were then liberated by rigorous pipetting and filtered through a strainer with a pore size of 100 μm. After centrifugation, the pellet was resuspended in culture medium and the muscle fibers transferred into 35 mm dishes coated with collagen type I (1 mg/ml) (BD). The culture medium consisted of DMEM/F12, 1% penicillin/streptomycin, 18% FBS, 10 ng/ml hEGF (Sigma), 1 ng/ml hbFGF (Sigma), 10 μg/ml human insulin (Sigma), and 0.4 μg/ml dexamethasone (Sigma) [29 (link)]. After 24 h, a fibroblast reduction step was performed by replating the cells. The cultured hMPCs were characterized as published before [14 (link), 29 (link)].