Human myoblasts from skeletal muscle biopsies: in vitro culture preparations for morphological and cytochemical analyses at light and electron microscopy

We describe protocols for the isolation of satellite cells from human muscle biopsies, for the in vitro culture of proliferating and differentiating myoblasts, and for the preparation of cell samples suitable for morphological and cytochemical analyses at light and electron microscopy. The procedures described are especially appropriate for processing small muscle biopsies, and allow obtaining myoblast/myotube monolayers on glass coverslips, thus preserving good cell morphology and immunoreactivity for protein markers of myoblast proliferation, differentiation, and senescence.These cell preparations are suitable for cytochemical, immunocytochemical, and FISH procedures at light microscopy, and can be observed not only in bright field, phase contrast, and differential interference contrast but also in fluorescence (which can hardly be used for cells grown on conventional plastic surfaces, which generally exhibit intense autofluorescence). In their ultrastructural cytochemical application, the protocols are intended for post-embedding techniques, by which ultrathin sections from a single sample may be used for detecting a wide variety of molecular markers