| Popis: |
Background: In skeletal muscle, 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. Even in substrates coated with proteins consisting of skeletal muscle-specific ECM compositions extracted from skeletal muscle, the function of each ECM protein remains, but the structural features of the basement membrane are lost. Therefore, in this study, we used decellularized muscle tissue and mouse myoblasts C2C12. We developed and evaluated a system to analyze the interaction between native ECM and myocytes. Methods: Chicken skeletal muscle was sliced into sheets and decellularized with 1% SDS solution, 0.1N NaOH solution, and 50% ethanol 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. Survival of myotubes in long-term culture was also confirmed by calcein staining. Results: Decellularization of sliced skeletal muscle created a flexible skeletal muscle ECM-derived culture substrate. C2C12 myoblasts adhered to scaffolds in the DSMS and developed adhesion plaques and filopodia. In addition, C2C12 myotubes showed orientation along with the ECM orientation within the DSMS. Furthermore, RT-PCR results showed that C2C12 myogenic differentiation was promoted on the DSMS since the expression levels of myoD, etc., were increased early. On the plastic dish, detachment of cells occurred from day 6 of differentiation, and few cells survived to day 12, but on DSMS, no detachment occurred even on day 12, allowing long-term culture.Conclusions: This study reports a novel myocyte culture method using sheet-like ECM structures obtained by decellularization. Myoblasts adhered to the sheet according to the orientation of the ECM, and the direction of myotube formation could be controlled. This culture technique reproduces a cell culture environment that reflects the properties of living skeletal muscle and allows studies on the interaction between the ECM and myocytes. |
