Soft Folding Skeletal Muscle Biobot Fabricated with a Conducting Hydrogel, PEDOT:PSS

Muscle for actuation in robotics, or biohybrid robotics, is a growing field combining biological and synthetic materials in centimeter-scale soft robots. While prior studies have leveraged the contraction of 2D cardiac monolayers to power swimming robots, culturing skeletal muscle in this form has proven challenging and has led to delamination of cells from their substrates due to the passive and active tension forces generated by skeletal muscle fibers during development and stimulated contraction. Given that skeletal muscle offers significant advantage over cardiac muscle in robotics, such as on/off control and adaptation to gain-of-function (exercise) and loss-of-function (damage) cues, there is a need to develop methods to control skeletal muscle contraction in 2D formats. We have developed a method for culturing C2C12 murine myoblast-derived muscle on a conducting polymer hydrogel, namely, poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate or PEDOT:PSS) coated with extracellular matrix hydrogels known to promote muscle differentiation, Matrigel and fibrin. These conductive hydrogels exhibit robust mechanical and electrical properties and enable targeted stimulation of skeletal muscle. Unlike previous studies in which electrical stimulation triggers contraction of entire tissues, our platform enables the control of muscle activation precisely and spatiotemporally in specific regions of the gel substrate. Our method of differentiating skeletal muscle on conductive substrates can be used as a building block to create complex, controllable soft robots.<br/>Since cell cultures with direct cell seeding on PEDOT led to minimal cell growth, we have developed a method to layer fibrin gel on top of PEDOT:PSS. PEDOT beams were sterilized and placed in growth medium for 1 day to drive swelling and protein adsorption that functionalized the gel surface for the binding of fibrin. Post swelling, a solution of 8mg/ml fibrinogen mixed with serum-free media, mixed with 4ul of thrombin was added to the PEDOT beams and left to polymerize with a weighted stamp to produce a thin gel layer. After the bilayer was created, 500,000 cells per well were seeded. After 2 days, growth medium was switched to differentiation medium and in 4 more days they were optically/electrically stimulated. Different concentrations of PEDOT:PSS were also tensile tested for stiffness prior. For concentrations of 2.3%, 3.5%, and 7%, preliminary Young’s Moduli were measured in the range of 200-800 kPa.<br/>Light vs electrical stimulation of optogenetic cells shows that both methods generate muscle contraction, indicating our ability to grow contractile muscle on PEDOT and maintain them in culture. Leveraging our open-source computational framework to map muscle displacement in 2D, we saw no significant difference in contractile strain (6-7%) between optical/electrical stim, indicating no negative effects of PEDOT on muscle function. While preliminary data shows no significant difference in contractility between stimulation setups, as might be expected given the conductivity of PEDOT, we predict studies where we align muscle fibers unidirectionally via grooved stamps on fibrin/PEDOT, and electrically stimulate directly through PEDOT, will provide a more controlled comparison. Concurrent with experiments, we’ve generated computational models for rapid <i>in silico </i>iteration of biohybrid robot design. Experimentally determined values of beam geometry, PEDOT/fibrin mechanical properties, and muscle force were input into our custom model. In our current configuration, the muscle layer applies a unidirectional shear force of 0.7 mN to the bilayer beam, generating a maximum deflection of 700 um, corresponding to about 5% strain in the muscle layer. This shows that the conducting substrate allows for significant beam deflection via muscle contraction, showcasing our platform’s ability to create soft robots powered by 2D skeletal muscle monolayers.