Dataset Information

Nanopattern surface improves cultured human myotube maturation.

ABSTRACT:

Background

In vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single-cell level.

Methods

Human primary myoblasts were cultured on 800-nm wide striated surface between two layers of Matrigel, and in a media supplemented with an inhibitor of TGFβ receptor. Gene expression, immunofluorescence, and Ca²⁺ measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes.

Results

We show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of α-actinin and STIM1, and occasionally ryanodine receptor 1. We also demonstrate that the myotubes present robust Ca²⁺ responses to repetitive electrical stimulations.

Conclusion

The present method describes a fast and efficient system to obtain well matured and functional myotubes in 2D culture allowing thorough analysis of single-cell Ca²⁺ signals.

SUBMITTER: Brunetti J

PROVIDER: S-EPMC8097894 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundSkeletal muscle contributes to roughly 40% of lean body mass, and its loss contributes to morbidity and mortality in a variety of pathogenic conditions. Significant insights into muscle function have been made using cultured cells, in particular, the C2C12 myoblast line. However, differentiation of these cells in vitro typically yields immature myotubes relative to skeletal muscles in vivo. While many efforts have attempted to improve the maturity of cultured myotubes, including the use of bioengineered substrates, lack of molecular characterization has precluded their widespread implementation. This study characterizes morphological, molecular, and transcriptional features of C2C12 myotubes cultured on crosslinked, micropatterned gelatin substrates fabricated using previously established methods and compares them to myotubes grown on unpatterned gelatin or traditional plasticware.MethodsWe used immunocytochemistry, SDS-PAGE, and RNAseq to characterize C2C12 myotubes grown on micropatterned gelatin hydrogels, unpatterned gelatin hydrogels, and typical cell culture substrates (i.e., plastic or collagen-coated glass) across a differentiation time course. The ability to form aligned sarcomeres and myofilament protein concentration was assessed. Additionally, the transcriptome was analyzed across the differentiation time course.ResultsC2C12 myotubes grown on micropatterned gelatin hydrogels display an increased ability to form aligned sarcomeres as well as increased contractile protein content relative to myotubes cultured on unpatterned gelatin and plastic. Additionally, genes related to sarcomere formation and in vivo muscle maturation are upregulated in myotubes grown on micropatterned gelatin hydrogels relative to control myotubes.ConclusionsOur results suggest that growing C2C12 myotubes on micropatterned gelatin hydrogels accelerates sarcomere formation and yields a more fully matured myotube culture. Thus, the use of micropatterned hydrogels is a viable and simple approach to better model skeletal muscle biology in vitro.