ECM components are essential for proper in vitro myogenesis

Abstract

Objective: In vitro models of skeletal muscle often utilize primary myoblast cells or myoblast cell lines. Myoblasts require adhesion to the extracellular matrix (ECM) to grow, proliferate, migrate, and differentiate in their natural environments in vivo. To meet the adhesion needs of adhesive cells under in vitro conditions, culture surfaces are coated with various biological or synthetic compounds. Within the scope of the study, the differentiation potential of H2K myoblasts, a cell line resembling primary myoblasts, were comparatively evaluated through morphological analysis on culture surfaces coated with various ECM and synthetic materials. Methods: The culture surfaces were coated with fibronectin and laminin, the major adhesion proteins of ECM; gelatin, a molecular derivative of collagen; matrigel, an ECM extract; and PLL, a synthetic poly-amino acid. Cells were allowed to differentiate in each culture medium for 4 days and their capacity to adhere to the surface and differentiation rates from myoblast to myotube were evaluated by morphological analysis. Results: In the uncoated culture environment, cells could only attach to 30-50% of the culture surface and myotube development was limited and not aligned with each other. On surfaces coated with PLL, no myotube development was observed and cells could only attach to 30-40% of the culture surface. Myotube development and alignment were similar on all surfaces coated with ECM components. On surfaces coated with ECM components laminin, fibronectin and matrigel, cells covered the entire culture surface and exhibited similar myotube development. However, on surfaces coated with gelatin, both cell adhesion to the surface and myotube development were limited compared to other ECM components. The mean myotube diameters of fibronectin, laminin, matrigel, PLL+laminin and gelatin were 49.71µm (±16.3µm), 52.31µm (±15.7µm), 51.9µm (±15.3µm), 53.06µm (±14.2µm) and 35.25µm (±11.4µm), respectively. Conclusion: Within the scope of the study, it was revealed that coating the culture surface with only a cationic material such as PLL does not support myogenesis and ECM components are needed for cell viability and differentiation.