Development of a primary skeletal muscle cell culture model from human muscle biopsies

Abstract

Purpose: This work aimed to develop and validate a culture model of primary human skeletal muscle cells. Methods: Muscle biopsies were obtained post-mortem from the vastus lateralis. The biopsies were enzymatically digested with collagenase 1A (1mg/mL) and dispase II (4,8mg/mL) for 60 minutes at 37°C in order to isolate fibroblasts and satellite cells. Then, the satellite cells (CD56+) were separated from fibroblasts (CD56-) by two methods: pre-plating or immunomagnetic cell sorting using an anti-CD56 antibody, a transmembrane glycoprotein expressed on the surface of satellite cells. Indeed, some human muscle biopsies contain a high proportion of fibroblasts that can contaminate the myoblasts cell culture if the separation method is not efficient. The performance of these cell separation methods was evaluated by FACS. We also compared the effects of two culture media on myoblasts proliferation (10%FBS vs. 10%FBS + 2%Ultroser G (UG)) using cell doubling time as parameter. Two differentiation media (2%FBS vs. 0.5%UG) were tested on the myogenic index, which corresponds to the proportion of cells that have fused to form myotubes. This index was determined through the detection of the myosin heavy chain (MYH) by immunofluorescence. Finally, we studied the expression of several muscle cell markers during myogenesis by qRT-PCR and immunofluorescence. Results: The cells obtained from the enzymatic digestion of the biopsy contained a varying proportion of CD56+ satellite cells (86,15 ± 12,09%; 95%CI:73,45 to 98,84). The cell population obtained after immunomagnetic cell sorting method contained more than 92% of CD56+ cells (96,11 ± 3,27%; 95%CI:92,68 to 99,54). In comparison, the ratio of CD56+ obtained by the preplating method was lower (84,19 ± 9,94%; 95%CI:73,76 to 94,62), but no significant difference was observed between both methods. The cell doubling time was significantly shorter with culture media containing 10%FBS and 2%UG than with the media containing only 10%FBS (25.70 ± 3,96h vs. 70.64 ± 21,68h, p = 0.0313). The myogenic index was slightly higher with the 0.5%UG than 2%FBS differentiating medium (62,75% ± 8,57% vs. 56,39 ± 16,45%). Finally, the increase of MyoD1, myogenin and MYH, three markers of differentiation, indicated the phenotypic transition of myoblasts to a myogenic phenotype. Conclusions: Immunomagnetic cell sorting allows better satellite cells and fibroblasts separation than the conventionally used preplating technique. Ultoser G serum substitute accelerates cell proliferation and improves the differentiation of myoblasts into myotubes. The markers MyoD1, myogenin and MYH are good markers of the myogenic differentiation of myoblasts. This in vitro model makes it possible to purify efficiently muscle satellite cells from human muscle biopsies. These cells can be used in in vitro studies to study the pathophysiological mechanisms of muscle disease and the mechanisms of action of treatments.