Development and maintenance of healthy muscle fibers rely on the myogenic potential of satellite cells (SC), muscle stem cells that proliferate and differentiate to form myotubes. Satellite cells are indispensable for post-hatch muscle growth as well as muscle repair and regeneration when myofibers are damaged. Pectoralis major of young broiler chicks (5-d olds) is a readily available source of SC, which can be used in vitro to elucidate cellular and molecular mechanisms responsible for muscle growth and regeneration in broilers. Here, we optimized a method for efficient isolation, purification, and differentiation of SC, from young broiler chicks. This procedure includes a simple method that allows SC to be purified from other muscle cell types that can impede the fidelity of follow-on experiments, particularly highly sensitive measures such as RNAseq. The methods for culturing and differentiating SC into multinucleated myotubes were also optimized by testing serum types, concentrations, and the effects of chicken embryo extract. Using the isolation procedure, a highly pure SC population (94.6 ± 2.11% Pax7+) with high viability and yield was obtained, and their capacity to differentiate into myotubes was confirmed. Enrichment for SC and myogenic capacity were maintained through multiple passages and after cryopreservation. Analysis of gene expression over the first 48 h of differentiation confirmed that SC exhibited the expected molecular signature of myogenesis. Taken together, this method simplifies the ability to isolate and maintain a relatively pure population of SC with strong myogenic potential from young broiler chicks, and should support downstream applications for assessing the impact of nutrients, metabolites, and other physiological cues on muscle growth and development in broilers.
Keywords: breast muscle; broiler; preplating; primary cell isolation; satellite cells.
The purpose of this study is to optimize the isolation and culture method for muscle stem cells, called satellite cells, in the breast muscle of meat-type chicken (Broiler). Satellite cells play pivotal roles in muscle development, growth, and muscle regeneration. One challenge of primary cell isolation is efficient and effective purification from a mixture of cell types, while maintaining viability and achieving high yield. Here, we developed a protocol enabling high yield and purity of primary satellite cells isolation and optimized culture conditions. We obtained about 95% purified satellite cells with high viability, and this method is simple and cost-effective compared to alternative cell sorting techniques that require cell-labeling steps and expensive equipment. Thus, this optimized satellite cell isolation and culture may be valuable for in vitro studies of cellular and molecular mechanisms responsible for muscle diseases (e.g., white striping and wooden breast) and the nutritional regulation of muscle development in broiler.
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