Isolation and ex vivo cultivation of single myofibers from porcine muscle
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Isolation and ex vivo cultivation of single myofibers from porcine muscle Katja Stange 1 & Hellen Elisa Ahrens 2 & Julia von Maltzahn 2 & Monika Röntgen 1 Received: 19 June 2020 / Accepted: 6 August 2020 / Published online: 22 September 2020 / Editor: Tetsuji Okamoto # The Author(s) 2020
Abstract 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. Keywords Porcine muscle fiber . Myofiber isolation . Nuclear density . Skeletal muscle . Pig
Introduction Studying skeletal muscle functionality in vitro is of utmost importance in order to elucidate cellular and molecular mechanisms controlling muscle development, maintenance, and metabolism, which also play a role in disease pathologies or tissue repair (Shefer and Yablonka-Reuveni 2005; Komiya et al. 2015; Stuelsatz et al. 2017). Myogenic stem cells, called satellite cells, were originally named by their localization between sarcolemma and basal lamina within their niche and are the main source for myonuclei (Mauro 1961). The myogenic cell population, including satellite cells and their progeny, extensively proliferates during early postnatal development and
* Monika Röntgen [email protected] 1
Institute of Muscle Biology and Growth, Growth and Development Unit, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
2
Research Group Stem Cells in Regeneration of Skeletal Muscle, Leibniz Institute on Aging, 07745 Jena, Germany
undergoes myogenic differentiation to enable muscle fiber growth (Swatland 1977; Campion et al. 1981; Miersch et al. 2017). Later on, adult satellite cells become
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