Isolation, Culture, and Differentiation of Bovine Muscle Resident Stem Cells.

Bovine muscle satellite cells (MuSC) and fibro-adipogenic progenitor cells (FAP) are muscle resident stem cells that are responsible for postnatal muscle growth, intramuscular fat deposition, and extracellular matrix generation. These cells are of increasing interest for the cultivated meat community due to their ability to generate all the major components of meat; additionally, these cells are of interest to conventional animal science research to elucidate mechanisms to improve meat quality. To use these cells for these goals, efficient and accurate cell isolation, culture, and differentiation are essential to evaluate their cell fate decisions and behaviors. In this protocol, we detail a simultaneous isolation of both MuSCs and FAPs with multiple intermediate stopping points, allowing for flexibility for day-of time constraints. We also detail improved growth conditions to maximize cell expansion and procedures to assess cell differentiation. This protocol provides a flexible isolation procedure that is compatible with sampling in modern slaughterhouses or from biopsies. Additionally, the differentiation procedures provide improved differentiation but still allow in vitro treatment and assessment. Key features • This protocol offers a flexible in-lab procedure to isolate bovine FAPs and MuSCs from tissue collected post-slaughter with multiple pause points. • The protocol demonstrates successful conditions to grow, expand, and differentiate bovine FAPs with an optimized adipogenic differentiation medium. • Strategies for planning your primary cell isolation, choosing the sampling location, and characterizing differentiation of bovine FAPs and MuSCs are included.