An Electrical Stimulation Culture System for Daily Maintenance-Free Muscle Tissue Production.

Low-labor production of tissue-engineered muscles (TEMs) is one of the key technologies to realize the practical use of muscle-actuated devices. This study developed and then demonstrated the daily maintenance-free culture system equipped with both electrical stimulation and medium replacement functions. To avoid ethical issues, immortal myoblast cells C2C12 were used. The system consisting of gel culture molds, a medium replacement unit, and an electrical stimulation unit could produce 12 TEMs at one time. The contractile forces of the TEMs were measured with a newly developed microforce measurement system. Even the TEMs cultured without electrical stimulation generated forces of almost 2 mN and were shortened by 10% in tetanic contractions. Regarding the contractile forces, electrical stimulation by a single pulse at 1 Hz was most effective, and the contractile forces in tetanus were over 2.5 mN. On the other hand, continuous pulses decreased the contractile forces of TEMs. HE-stained cross-sections showed that myoblast cells proliferated and fused into myotubes mainly in the peripheral regions, and fewer cells existed in the internal region. This must be due to insufficient supplies of oxygen and nutrients inside the TEMs. By increasing the supplies, one TEM might be able to generate a force up to around 10 mN. The tetanic forces of the TEMs produced by the system were strong enough to actuate microstructures like previously reported crawling robots. This daily maintenance-free culture system which could stably produce TEMs strong enough to be utilized for microrobots should contribute to the advancement of biohybrid devices.