Skeletal muscle is a postmitotic tissue type which retains the capacity to regenerate due to the activation of quiescent precursor cells, the satellite cells. Nonetheless, an ex vivo enrichment of skeletal muscle cells is hampered by fibroblast overgrowth. Here we have tested the effect of in vitro irradiation on the yield of ex vivo enriched skeletal muscle cells. Hind limb muscles of mouse embryos (E 18.5) were harvested, isolated enzymatically and grown in culture, resulting in a mixed population of attaching and proliferating cells. Immunohisto-chemistry demonstrated that after 48 hours up to 50% of the cells were myogenic precursors. While pax7-positive satellite cells were still proliferating, desmin-positive myoblasts differentiated and fused to form multinucleated myotubes. On day 9, the rapidly dividing fibroblast population overgrew the myogenic cells. This effect was eliminated using a single irradiation of 15 Gy to the primary satellite cell culture at day 3. While this led to upregulation of p53 and fragmentation of nuclei in non-myogenic cells, muscle cell enrichment and formation of multinucleated myotubes were observed. The spontaneously beating myotubes could be held in culture for more than 40 days.

We conclude that murine satellite cells are highly resistant to irradiation and that this leads to enhanced myogenesis ex vivo.