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

Abstract

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotube 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 on the groove patterns with different widths (from 0.4 to 200 μm), we observed an enhanced chiral orientation of cells developed on wide grooves (50 and 100 μm of width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, those 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 numtatics of cells for myogenesis. Finally, by arranging wide groove/stripe patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes are formed, providing a strategy for engineering skeletal muscle tissue formation.

UV laser-processed microstructure for building biohybrid actuators with anisotropic movement

“…These behaviors of myotube alignment according to curvature are considered to depend on the structural stability between Cir-MGs and Cur-MGs. Moreover, similar to previous reports that concentric myotube alignment along MGs was not observed at curvatures higher than 1 mm −1 [52], myotubes on Cir-MGs did not align at curvatures higher than 2 mm −1 . These results indicate that the degree of myotube curvature is controlled by the curvature of the MG, which acts as a scaffold, thereby yielding a threshold of curvature for the alignment of myotubes.…”

“…Recently, the alignment of myotubes by placing a series of S-MGs in a curvilinear arrangement was reported [52]; however, there are few reports of myotubes aligned in a curve by a curved MG. Therefore, the effect of curvature in Cur-MGs on myotube alignment was examined using Cur-MGs fabricated by the UV laser system.…”

Straightforward Cell Patterning with Ultra‐Low Background Using Polydimethylsiloxane Through‐Hole Membranes

“…Also, this high efficient cell patterning with ultra-low background and high fidelity was not the case for the reported methods, which commonly solved one of these two key elements. [7,18,19,26,27] Furthermore, other patterning results about customizable cell arrangements were obtained too (Figure 6 and S6, Supporting Information). As illustrated in Figure 6A, circular cell spots can be intentionally arranged in different letters that were "C", "S", and "U".…”

“…Also, this high efficient cell patterning with ultra‐low background and high fidelity was not the case for the reported methods, which commonly solved one of these two key elements. [ 7,18,19,26,27 ]…”

“…[13][14][15] Comparatively, the micro/nano-topographical or biochemical-molecule-modified interfaces enable cells to adhere to the pre-defined regions. [16][17][18][19] The patterning definition is quite optional and the patterning diversity can be effectively guaranteed based on the established printing methods. Various other basic geometry units (e.g., triangle, star, diamond, anchor, arrow) and arrangements (e.g., smiling face, different words, and numbers) of cell patterns were generated as needed.…”