In vitro human skeletal muscle platform for the evaluation of functional outcomes

Resumen

Skeletal muscle is a striated tissue whose main function is to produce the force needed to support locomotion, posture, respiration, and other metabolic processes. Nowadays, most in vitro myotube cultures do not fully recapitulate muscle characteristics, and therefore, these studies are not physiologically relevant to human muscle. This work presents a new experimental approach to achieve a high-throughput platform of 2D mature functional myotubes. First, cell cultures were grown onto PDMS-grooves (μGrooves) on a glass substrate. Different sizes and shapes were tested to confine the myoblasts and promote their alignment within the micro-well. Electrical stimulus (4V, 6ms, 0.1Hz) was applied for 24 hours to increase myotube differentiation and maturation. Myotubes were tested for orientation, maturation, fusion index, cell density, calcium handling, and, contractile function. Our results demonstrate the capability of this platform to obtain highly homogeneous cultures composed of individual myotubes. These cultures replicate native characteristics of the skeletal muscle regarding excitationcontraction capacity and appropriate responsiveness to different stimuli. Furthermore, our protocol minimizes the variability of maturation and cytosolic calcium levels within cultures, which are significant limitations of traditional myotube cultures. In this regard, we found that μGrooves reduced the coefficient of variation of intracellular calcium levels. Therefore, this platform enables robust high content assays of calcium levels and contractile readouts with increased sensitivity compared to standard cultures. Finally, in addition to mechanistic studies, this platform could be used in combination with myotubes from patients with different muscle pathologies for high throughput drug screening assays.