Evaluation of material effects on three-dimensional cultured skeletal muscle cells for biohybrid robots

Robots are traditionally confined to controlled environments such as factories, where human interactions are limited. However, the demand for robots that are capable of collaborating with humans is increasing. To achieve symbiosis, integrating the physical flexibility and environmental adaptability of living organisms into robotic systems is crucial. An example of such a robot is a biohybrid robot driven by three-dimensional (3D) cultured skeletal muscle cells. These muscle cells, which are composed of myoblasts and an extracellular matrix (ECM), contract and generate force in response to external stimuli. The standardization of such 3D-cultured skeletal muscle cells is essential for practical applications. However, their complete standardization has not yet been achieved. The contractile force of 3D-cultured skeletal muscle cells produced via 3D printing is still insufficient for practical applications as actuators in biohybrid robots. In a previous study, we developed a simple fabrication method for 3D-cultured skeletal muscle cells. These bio-cultured artificial muscle (BiCAM) cells can control the shape and cell alignment of tissues. Differences in the composition of an ECM have been suggested to affect the contractile force of 3D skeletal muscle tissues; however, their impact on the response characteristics remains poorly understood. In this study, we investigated how the ECM composition influences the contractile force of 3D skeletal muscle cells in biohybrid robots as a step toward their eventual standardization. Compared with tissues cultured under MF conditions, in which electrically induced contraction was previously confirmed, tissues cultured under CM conditions exhibited an approximately two-fold greater contractile force at voltage amplitudes of 10 and 30 V. Furthermore, the fabrication success rate was 100 $\%$ under CM conditions but only 62.5-70 $\%$ under other ECM conditions. In contrast, although CM tissues generated larger forces, tissues cultured under MgF and CMg conditions exhibited higher-frequency response. These findings demonstrated that the BiCAM is a viable actuator and offers new possibilities for the design of biohybrid robots.