Nakada Satoshi, Yamashita Yuri, Akiba Seiya, Shima Takeru, Arikawa-Hirasawa Eri
Japanese Center for Research on Women in Sport, Juntendo University Graduate School of Health and Sports Science, Chiba 270-1695, Japan.
Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
Bioengineering (Basel). 2022 Jul 12;9(7):309. doi: 10.3390/bioengineering9070309.
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.
在骨骼肌中，肌纤维高度有序地排列并包裹在基膜内。包括各种细胞外基质（ECM）蛋白在内的几种微加工底物模型未能模拟天然肌肉的宏观结构。因此，我们开发并评估了一种使用脱细胞肌肉组织和小鼠成肌细胞C2C12的系统，以分析天然ECM与心肌细胞之间的相互作用。将鸡骨骼肌切成薄片并进行脱细胞处理，以制备脱细胞骨骼肌片（DSMS）。然后将C2C12接种在DSMS上并进行分化。进行ECM分子的免疫染色，以检查成肌细胞粘附状态、肌管取向和IV型胶原取向之间的关系。通过钙黄绿素染色确认长期培养中肌管的存活。C2C12成肌细胞粘附于DSMS中的支架上，并形成粘着斑和丝状伪足。此外，C2C12肌管在DSMS内沿ECM取向显示出取向。与塑料培养皿相比，DSMS上发生脱离的可能性较小，并且可以进行长期孵育。这种培养技术再现了反映活体骨骼肌特性的细胞培养环境，从而允许对ECM与心肌细胞之间的相互作用进行研究。
