Improved Tissue Culture Conditions for Engineered Skeletal Muscle Sheets

Engineered skeletal muscle is a promising therapeutic substitute for injured or disease tissue.  Unfortunately, the potential clinical utility of engineered muscle is currently restricted by the limited in vitro capacity of expanded muscle precursor cells to fuse and form mature myofibers.  Objective: The purpose of this study was to use an engineered isotropic skeletal muscle sheet to explore the impact of 1) cocultured fibroblasts and 2) fibroblast conditioned media (fCM) on in vitro myogensis. Methods: In this study, muscle sheets were prepared by seeding varying ratios of a7-positive nSKM cells and a7-negative fibroblasts on a biomimetic extracellular matrix (ECM) and culturing the resulting tissue in either control media (10% HS DMEM) or fCM. The tissue sheets were evaluated for myofiber density at three time points. Results: A high purity muscle precursor cell population was successfully collected from a mixed population of rat nSKM cells via cell sorting for positive a7 integrin expression. After two days in culture, multinucleated myofibers were observed in all experimental groups.  On day 6 and 8, the tissue sheets with low fibroblast concentrations (0, 10, 15%) cultured in fCM had an increased average myofiber density (4.8±0.2 myofibers/field) compared to tissue sheets with high fibroblast concentrations (50, 75%) cultured in control media (1.0±0.1 myofibers/field). In addition to higher myofiber density, fCM promoted longer and thicker myofibers, indicative of a more mature phenotype. Conclusions: Paracrine factors released by fibroblasts may represent an important target for potentiating myogenesis in engineered skeletal muscle constructs.