Scalable pattern formation of skeletal myotubes by synergizing microtopographic cues and chiral nematics of cells.

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotubes deviating from the groove boundaries is commonly observed but has long been unattended. In this study, we report a method to guide the formation of skeletal myotubes into scalable and controlled patterns. By inducing C2C12 myoblasts onto grooved patterns with different widths (from 0.4 to 200 μ m), we observed an enhanced chiral orientation of cells developing on wide grooves (50 and 100 μ m width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of the cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, these chiral myotubes were formed with enhanced length, diameter, and contractility on wide grooves. Treatment of latrunculin A (Lat A) suppressed the chiral rotation and migration of cells as well as the myotube formation, suggesting the essence of chiral nematics of cells for myogenesis. Finally, by arranging wide grooved/striped patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes were formed, providing a strategy for engineering skeletal muscle tissue formation.