Impact of Cell Seeding Density and Cell Confluence on Human Tissue Engineered Skeletal Muscle

Tissue engineering methodologies have the potential to treat volumetric muscle loss via the growth of exogenous skeletal muscle grafts from small autogenous muscle biopsies. A significant obstacle preventing the widespread use of engineered skeletal muscle grafts in a clinical setting is the high number of skeletal muscle stem cells, known as satellite cells, required for fabrication of human-sized skeletal muscle tissue. Additionally, there is a lack of work adapting engineered constructs created for animal models into skeletal muscle engineered from a primary human skeletal muscle cell source. For this study, we used scaffold-free tissue-engineered skeletal muscle units (SMUs) to determine the impact of cell seeding density on the ability to fabricate functional human engineered skeletal muscle. Following established protocols, human skeletal muscle isolates were cultured into SMUs at five different cell seeding densities: 1000, 2500, 5000, 10,000, and 25,000 cells/cm². Following previous human SMU work, SMUs prepared at a cell seeding density of 10,000 cells/cm² served as controls. Additionally, the impact of cell monolayer confluency on the outcome of human cell-sourced SMU fabrication was investigated at both the 1000 and 10,000 cells/cm² seeding densities. Light microscopy was used to examine myotube formation and hypertrophy in cell monolayers. After the formation of three-dimensional constructs, SMUs underwent maximum tetanic isometric force production measurements and immunohistochemical staining to examine SMU contractile function and muscle-like structure, respectively. Results indicate that the 25,000 cells/cm² cell seeding density was detrimental to the contractile function of human cell-sourced SMUs, which had significantly lower maximum tetanic forces compared with SMUs seeded at lower densities. Compared with control, low cell seeding densities (1000-5000 cells/cm²) have no detrimental impact on SMU skeletal muscle growth, maturation, or contractility. Cell cultures seeded at 1000 cells/cm² and allowed to proliferate to 90-100% confluency before treatment in muscle differentiation media (MDM) resulted in SMUs with greater contractile forces and total muscle structure compared with cell cultures switched to MDM when underconfluent or overconfluent. In conclusion, initial cell seeding density for SMU fabrication can be decreased to as low as 1000 cells/cm² without negatively impacting SMU muscle-like structure and function. Impact Statement Our research suggests that during the translation of skeletal muscle tissue engineering technologies from animal to human cell sources, initial starting cell seeding density can be significantly lowered without negatively impacting engineered skeletal muscle growth, maturation, or contractile function. Decreasing the initial cell density, and, thus, the muscle biopsy size required to fabricate an engineered human skeletal muscle, increases the potential for the clinical adoption of tissue-engineered based therapies for volumetric muscle loss.