Sheep satellite cells are more similar to human satellite cells regarding metabolism, life span, proliferation, and differentiation, than satellite cells of the rat and mouse. Regeneration of skeletal muscle in response to injury occurs through the fusion of a population of stem cells, known as satellite cells, with injured myofibers. Satellite cells are widely used in the modeling and treatment of diseases like heart insufficiency, neurological diseases, muscular dystrophy, cerebral cell transplantation for the treatment of migraines, screening, and the production of new drugs. This study was performed to describe the necessary methods to isolate and culture of primary satellite cells (PSCs) obtained from a 50 to 60-day-old sheep fetus, single-cell cloning of transfected PSCs and used for plotting the growth curve, mycoplasma and bacterial presence tests. Skeletal muscle tissues of hind-limbs were collected from sheep fetuses obtained from a local abattoir. After enzymatic digestion, a combination of cells, including myogenic satellite cells and non-myogenic cells were cultured in the culture flask. Flasks were replaced after 3 hours to isolate non-myogenic cells, such as fibroblasts. After six days, the cells were differentiated to myoblasts. Using a differentiation medium containing horse serum, myotubes cells were observed on the flask, indicating that the cultured cells were satellite cells. The mRNA expression of the PAX7 gene was used to confirm the presence of satellite cells. Also, the results showed that satellite cells were grown in a medium culture containing 5% FBS without differentiation and required 10% FBS to initiate differentiation.

Keywords