Leveraging microtopography to pattern multi-oriented muscle actuators

Engineering skeletal muscle tissue with precisely defined alignment is of significant importance for applications ranging from drug screening to biohybrid robotics. Aligning 2D contractile muscle monolayers, which are compatible with high-content imaging and can be deployed in planar soft robots, typically requires micropatterned cues. However, current protocols for integrating microscale topographical features in extracellular matrix hydrogels require expensive microfabrication equipment and multi-step procedures involving error-prone manual handling steps. To address this challenge, we present STAMP (simple templating of actuators <i>via</i> micro-topographical patterning), an easily accessible and cost-effective one-step method to pattern microtopography of various sizes and configurations on the surface of hydrogels using reusable 3D printed stamps. We demonstrate that STAMP enables precisely controlling the alignment of mouse and human skeletal muscle fibers without negatively impacting their maturation or function. To showcase the versatility of our technique, we designed a planar soft robot inspired by the iris, which leverages spatially segregated regions of concentric and radial muscle fibers to control pupil dilation. Optogenetic skeletal muscle fibers grown on a STAMPed iris substrates formed a multi-oriented actuator, and selective light stimulation of the radial and concentric fibers was used to control the function of the iris, including pupil constriction. Computational modeling of the biohybrid robot as an active bilayer matched experimental outcomes, showcasing the robustness of our STAMP method for designing, fabricating, and testing planar biohybrid robots capable of complex multi-DOF motion.