High-Throughput Label-Free Continuous Quantification of Muscle Stem Cell Proliferation and Myogenic Differentiation.

BACKGROUND: Quantifying muscle satellite cell proliferation and differentiation is crucial for applications in muscle regeneration, disease modeling, and cultivated meat research. Traditional fluorescence-based assays, while sensitive, are labor-intensive, endpoint-restricted, and disruptive to myotube integrity. METHODS: In this study, we present a novel high-contrast brightfield (HCBF) imaging technique for high-throughput, label-free assessment of both satellite cell proliferation and myogenic differentiation. Using the BioTek Cytation 5 automated imager and Gen5 software (Agilent Technologies), we optimized imaging parameters to achieve continuous, highly time-resolved quantification in standard 96- and 384-well formats without any additional reagents or cell manipulation needed. RESULTS: Our approach enabled detailed kinetic profiling of satellite cell behavior, revealing myotube formation dynamics, species-specific media responses, optimal seeding conditions and the influence of mechanical factors on differentiation. We also demonstrated that serum-free media formulations could support efficient myotube formation in both bovine and porcine satellite cells, while having very different myotube kinetics and morphology than serum-containing samples. Furthermore, we highlighted the high degree of well-to-well variation and the sporadic formation and detachment of myotubes in culture, and the interesting phenomena of a second wave of myotubes being formed following detachment in serum-containing samples. Additionally, the 384-well format enabled a label-free screening method to assess clonal myogenicity of isolated satellite cells. CONCLUSION: By eliminating the need for genetic labeling, invasive staining or specialized consumables, our high-throughput HCBF methodology advances myogenic research, offering new opportunities for efficient screening and highly detailed kinetic data acquisition for serum-free media development, drug discovery and pathophysiological testing for both cultivated meat and musculoskeletal research.