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Myocyte Culture with Decellularized Skeletal Muscle Sheet with Observable Interaction with the Extracellular Matrix.

ABSTRACT: In skeletal muscles, muscle fibers are highly organized and bundled within the basement membrane. Several microfabricated substrate models have failed to mimic the macrostructure of native muscle, including various extracellular matrix (ECM) proteins. Therefore, we developed and evaluated a system using decellularized muscle tissue and mouse myoblasts C2C12 to analyze the interaction between native ECM and myocytes. Chicken skeletal muscle was sliced into sheets and decellularized to prepare decellularized skeletal muscle sheets (DSMS). C2C12 was then seeded and differentiated on DSMS. Immunostaining for ECM molecules was performed to examine the relationship between myoblast adhesion status, myotube orientation, and collagen IV orientation. Myotube survival in long-term culture was confirmed by calcein staining. C2C12 myoblasts adhered to scaffolds in DSMS and developed adhesion plaques and filopodia. Furthermore, C2C12 myotubes showed orientation along the ECM orientation within DSMS. Compared to plastic dishes, detachment was less likely to occur on DSMS, and long-term incubation was possible. This culture technique reproduces a cell culture environment reflecting the properties of living skeletal muscle, thereby allowing studies on the interaction between the ECM and myocytes.

SUBMITTER: Nakada S

PROVIDER: S-EPMC9311603 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.

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Myocyte Culture with Decellularized Skeletal Muscle Sheet with Observable Interaction with the Extracellular Matrix.

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