Isolation and ex vivo cultivation of single myofibers from porcine muscle

The isolation and cultivation of intact, single myofibers presents a superior approach for studying myogenic cells in their native position. The cells’ characteristics remain more similar to muscle tissue than in cell culture. Nevertheless, no routinely used method in higher vertebrates exists. Therefore, we aimed at establishing the isolation and cultivation of single myofibers from porcine muscle. For the first time, we implemented the isolation of intact myofibers from porcine fibularis tertius muscle by enzymatic digestion and their subsequent cultivation under floating conditions. Confocal microscopy showed intact myofibrill structures in isolated myofibers. Myogenic cells were able to proliferate at their parent myofiber as shown by the increase of myonuclear number during culture. Additionally, the described method can be used to investigate myogenic cells migrated from isolated myofibers. These cells expressed myogenic markers and were able to differentiate. In the future, our method can be used for genetic manipulation of cells at myofibers, investigation of growth factors or pharmacological substances, and determination of interactions between myofibers and associated cells. Working with isolated myofibers has the potential to bridge conventional cell culture and animal experiments. Adapting the method to porcine muscle allows for application possibilities in veterinary medicine as well as in biomedical research, which cannot be addressed in rodent model systems.