Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice

Abstract

Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

Combined substrate micropatterning and FFT analysis reveals myotube size control and alignment by contact guidance

“…The images illustrate the polarised actin filaments in the cells (left image); Also, (right image) the myoblast cells located in close proximity exhibit a characteristic sarcomeric actin patterning indicating the differentiating status of the myoblasts (Duan & Gallagher, 2009). Simple applicability of the technique has made it method of choice in comparison to more complex and expensive options such as fluorescence activated cell sorting (Hindi et al, 2017). Thus, we observed that differential adherence technique enabled the selection of myoblasts.…”

“…Both the cellular markers were traced in culture for 6 days in vitro (DIV) through fluorescence microscopy. About 74 ± 3.3 percentage of the cells were stained positive for Pax7, a marker for proliferating myoblasts (Hindi, McMillan, Afroze, Hindi, & Kumar, 2017;Seale et al, 2000).…”

“…Thus, we observed that differential adherence technique enabled the selection of myoblasts. Simple applicability of the technique has made it method of choice in comparison to more complex and expensive options such as fluorescence activated cell sorting (Hindi et al, 2017). Figure 2a shows the morphological changes in the cells at various stages of culture in vitro.…”

Efficient and high yield isolation of myoblasts from skeletal muscle

“…Although these cells can be isolated directly from the mononuclear cell population by enzymatic digestion and titration of skeletal muscle tissue,(Rando and Blau, 1994; Hindi et al, 2017) the efficiency of this method is low due to the limited number of satellite cells released from the skeletal muscle niche (1–2 × 10 5 myoblasts from the hindlimb muscles of one adult mouse (Yi and Rossi, 2011, Motohashi et al, 2014)). Furthermore, these protocols require passing the muscle slurry through 40 or 70 µm cell strainer, eliminating progenitor cells that may remain bonded to the myofibers (Danoviz and Yablonka-Reuveni, 2012).…”

“…Furthermore, these protocols require passing the muscle slurry through 40 or 70 µm cell strainer, eliminating progenitor cells that may remain bonded to the myofibers (Danoviz and Yablonka-Reuveni, 2012). The majority of cells isolated using these methods are fibroblasts and other non-myogenic cell types, necessitating further purification steps by applying several rounds of pre-plating and attachment of fibroblasts to collagen coated surface (Hindi et al, 2017), fluorescence-activated cell sorting (FACS) (Yi and Rossi, 2011) or magnetic-activated cell sorting (MACS) (Motohashi et al, 2014). …”