Soft Perfusable Device to Culture Skeletal Muscle 3D Constructs in Air

Devices for in vitro culture of three-dimensional(3D) skeletal muscle tissues have multiple applications, includingtissue engineering and muscle-powered biorobotics. In both cases,it is crucial to recreate a biomimetic environment by using tailoredscaffolds at multiple length scales and to administer prodifferentiativebiophysical stimuli (e.g., mechanical loading). Onthe contrary, there is an increasing need to develop flexible biohybridrobotic devices capable of maintaining their functionality beyondlaboratory settings. In this study, we describe a stretchable andperfusable device to sustain cell culture and maintenance in a 3Dscaffold. The device mimics the structure of a muscle connected totwo tendons: Tendon-Muscle-Tendon (TMT). The TMT deviceis composed of a soft (E & SIM; 6 kPa) porous (porediameter: & SIM;650 & mu;m) polyurethane scaffold, encased withina compliant silicone membrane to prevent medium evaporation. Two tendon-likehollow channels interface the scaffold with a fluidic circuit anda stretching device. We report an optimized protocol to sustain C2C12adhesion by coating the scaffold with polydopamine and fibronectin.Then, we show the procedure for the soft scaffold inclusion in theTMT device, demonstrating the device's ability to bear multiplecycles of elongations, simulating a protocol for cell mechanical stimulation.By using computational fluid dynamic simulations, we show that a flowrate of 0.62 mL/min ensures a wall shear stress value safe for cells(<2 Pa) and 50% of scaffold coverage by an optimal fluid velocity.Finally, we demonstrate the effectiveness of the TMT device to sustaincell viability under perfusion for 24 h outside of the CO2 incubator. We believe that the proposed TMT device can be consideredan interesting platform to combine several biophysical stimuli, aimedat boosting skeletal muscle tissue differentiation in vitro, opening chances for the development of muscle-powered biohybridsoft robots with long-term operability in real-world environments.