Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
Skelet Muscle. 2021 May 5;11(1):12. doi: 10.1186/s13395-021-00268-3.
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.
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 Ca measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes.
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 Ca responses to repetitive electrical stimulations.
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 Ca signals.
在体外使用 2D 培养来成熟原代人肌母细胞仍然是一个具有挑战性的过程，会导致不成熟的纤维，内部组织和功能较差。然而，这将代表一个有价值的系统，可以在单细胞水平上从患者肌母细胞研究肌肉生理学或病理生理学。
人原代肌母细胞在两层 Matrigel 之间的 800nm 宽的条纹表面上培养，并在补充有 TGFβ 受体抑制剂的培养基中培养。在成熟过程中的各个时间点进行基因表达、免疫荧光和电刺激后的 Ca 测量，以评估肌管的组织和功能。
我们表明，在培养 10 天后，肌管显示出许多功能性乙酰胆碱受体簇，并表达肌球蛋白重链和二氢吡啶受体的成人同工型。此外，肌管内部组织良好，具有α-辅肌动蛋白和 STIM1 的条纹，偶尔还有 Ryanodine 受体 1。我们还证明，肌管对重复电刺激有很强的 Ca 反应。
本方法描述了一种快速有效的 2D 培养方法，可获得成熟且功能良好的肌管，允许对单细胞 Ca 信号进行彻底分析。
