Development of an in vitro platform for the analysis of contractile and calcium dynamics in single human myotubes

outcomes. To promote maturation and decrease heterogeneity, we have incorporated engineered structures into myotube cultures, based on a PDMS thin layer with micrometer-sized grooves (μGrooves) placed over a glass substrate. Different sizes and shapes of μGrooves were tested for their ability to promote alignment and fusion of myoblasts and enhance their differentiation into myotubes. A 24 hour electrical field stimulation protocol (4 V, 6 ms, 0.1 Hz) was used to further promote myotube maturation, after which several myotube features were assessed, including myotube alignment, width, fusion index, contractile function, and calcium handling. Our results indicate superior calcium and contractile performance in μGrooved myotubes, particularly with the 100 μm-width 700 μm-long geometry (7 : 1). This platform generated homogeneous and isolated myotubes that reproduced native muscle features, such as excitation-contraction coupling and force-frequency responses. Overall, our 2D muscle platform enables robust high-content assays of calcium dynamics and contractile readouts with increased sensitivity and reproducibility compared to traditional myotube cultures, making it particularly suitable for screening therapeutic candidates for different muscle pathologies.