Poster U15 - A versatile method for patterning complex multi-oriented muscle architectures from mouse and human myoblasts

Introduction: Skeletal muscle is a key player of the locomotor system. Reduced muscle contractility is associated with diseases like Duchenne muscular dystrophy and amyotrophic lateral sclerosis. In vitro muscle models producing physiologically relevant forces are valuable tools to explore new therapeutic strategies. Skeletal muscle is also an excellent candidate actuator for soft robotics. However, these applications are limited by the difficulty of controlling muscle fiber alignment in vitro. For example, random alignment reduces the comparability of net forces generated by different biological replicates in therapeutic screens. Controlling muscle orientation would also permit complex motions for robotics, like actuating circular valves. Existing strategies to align muscle tissue often rely on microfabricated scaffolds that impair microscopy, in addition to requiring complex fabrication protocols. Alternative methods in which proteins are patterned on stiff abiotic substrates lack important cues from the extracellular matrix, and ultimately result in muscle delamination. Directly patterning grooves in hydrogels overcomes these limitations. However, current protocols are tedious and comprise manual handling steps, which limits their accessibility and yield.

Materials and

Methods: To address these issues, we developed a simple hydrogel molding approach to generate 2.5D sheets of aligned, contractile muscle in a variety of culture formats and from different cell sources (Figure 1a). We used 3D-printed stamps to pattern microscopic ridges into a fibrin gel layer cast in a 24 well plate, after optimizing our mold release and gel loading strategy. We designed the grooves so that their widths and heights correspond to 1x, 2x, 5x and 10x the size of single myoblasts in suspension. We then seeded myoblasts from murine and human origin on those micropatterned scaffolds, differentiated them into muscle fibers, and assessed their alignment as a function of groove size.

Results, Conclusions, and Discussions: Our ongoing work suggests that grooves with dimensions comparable to the size of single cells yield the most aligned muscle fibers, irrespective of cell source, likely by constraining the direction of growth and fusion of myoblasts. On the other hand, the orientation of myofibers grown on non-patterned scaffolds varies throughout the well (Figures 1b and 1c). All muscle tissues responded to electrical stimulation by contracting along the direction of fiber alignment, as quantified with our custom computational framework enabling to convert videos of muscle contraction into spatial maps of force production. Next, we applied our method for integrating microscale guidance cues to different culture formats and complex geometries. We generated aligned muscle in a 3D-printed microfluidic channel that would be amenable to cell culture under flow conditions (Figure 1d). In addition, we fabricated a fibrin scaffold patterned with a series of concentric and radial microgrooves arranged around a circular hole, mimicking the ocular muscles and the pupil. Murine muscle cells – which had been engineered beforehand to express light-sensitive channelrhodopsin – grew and differentiated in a multi-layered ring configuration (Figure 1e). We controlled the contraction of the concentric and radial layers separately by selectively shining blue light on either region, thereby reproducing how the iris controls the opening and closing of the pupil. Overall, our method provides the community with a versatile way of introducing microscale topographical cues in extracellular matrix hydrogels. In particular, it allows to generate complex multi-oriented structures, such as muscle fibers aligned in a radial, concentric or pennate orientation, unlike existing strategies that were limited to linear alignment. Furthermore, our method could be readily extended to patterning tissues beyond muscle, thereby providing the tissue engineering community with a simple and versatile method to control cellular orientation.

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