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

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.

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.

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.

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.