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

Satoshi Nakada; Yuri Yamashita; Seiya Akiba; Takeru Shima; Eri Arikawa-Hirasawa

doi:10.3390/bioengineering9070309

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

Bioengineering (Basel). 2022 Jul 12;9(7):309. doi: 10.3390/bioengineering9070309.

Authors

Satoshi Nakada¹, Yuri Yamashita^{2

3

4}, Seiya Akiba², Takeru Shima^{2

5}, Eri Arikawa-Hirasawa^{1

2

3

4}

Affiliations

¹ Japanese Center for Research on Women in Sport, Juntendo University Graduate School of Health and Sports Science, Chiba 270-1695, Japan.
² Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
³ Aging Biology in Health and Disease, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
⁴ Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
⁵ Faculty of Education, Gunma University, Maebashi 371-8510, Japan.

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.

Keywords: cell culture; decellularization; extracellular matrix; microdevice; myoblasts; skeletal muscle.

Grants and funding

This research was funded by a Grant-in-Aid from JSPS KAKENHI (grant numbers 21K17589 and 19K19951) and the Acceleration Program for Intractable Diseases Research Utilizing Disease-specific iPS Cells (20bm0804005h0004), which were granted by the Japan Agency for Medical Research and Development, Japanese Society for the Promotion of Science, Grants-in-Aid of Research into Rare and Intractable Diseases (H29-nanchitou(nan)-ippan-030) from the Ministry of Health, Labour, and Welfare of Japan, and an Intramural Research Grant (2–5) for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry.