Akiyama Yoshitake, Nakayama Akemi, Nakano Shota, Amiya Ryuichiro, Hirose Jun
Faculty of Textile Science and Engineering, Shinshu University, 3-15-1 Tokida, Ueda, Nagano, Japan.
Department of Biomedical Engineering, Shinshu University, 3-15-1 Tokida, Ueda, Nagano, Japan.
Cyborg Bionic Syst. 2021 Apr 8;2021:9820505. doi: 10.34133/2021/9820505. eCollection 2021.
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.
低劳动强度生产组织工程肌肉（TEMs）是实现肌肉驱动装置实际应用的关键技术之一。本研究开发并展示了一种具备电刺激和培养基更换功能的免日常维护培养系统。为避免伦理问题，使用了永生化成肌细胞C2C12。该系统由凝胶培养模具、培养基更换单元和电刺激单元组成，一次可生产12个TEMs。用新开发的微力测量系统测量TEMs的收缩力。即使在无电刺激条件下培养的TEMs也能产生近2 mN的力，强直收缩时缩短10%。关于收缩力，1 Hz单脉冲电刺激最为有效，强直收缩时的收缩力超过2.5 mN。另一方面，连续脉冲会降低TEMs的收缩力。苏木精-伊红（HE）染色的横截面显示，成肌细胞主要在周边区域增殖并融合成肌管，内部区域细胞较少。这一定是由于TEMs内部氧气和营养物质供应不足所致。通过增加供应，单个TEM或许能够产生高达约10 mN的力。该系统生产的TEMs的强直收缩力足以驱动如先前报道的爬行机器人等微结构。这种能够稳定生产足够强大以用于微型机器人的TEMs的免日常维护培养系统应有助于生物混合装置的发展。
