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

Abstract

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.

Contractile force assessment methods for in vitro skeletal muscle tissues

“…Electrical stimulation is the most frequently used mechanism to induce CF in engineered muscles by mimicking motoneuron activity on these tissues ( Cheng et al, 2014b ; Rangarajan et al, 2014 ; Ikeda et al, 2016 ; Akiyama et al, 2021 ). It has also been used during muscle development to improve muscle size, sarcomere formation, Ca 2+ transients, and CF production ( Park et al, 2008 ; Langelaan et al, 2011 ; Khodabukus et al, 2019 ).…”

“…In 2D models, CSA can be estimated as a circular shape, or calculated as an elliptical shape from the thickness and the width of the myotubes ( Pirozzi et al, 2013 ; Jeon et al, 2019 ). As for 3D constructs, the CSA has been approximated to circular ( Hinds et al, 2011 ; Juhas and Bursac, 2014 ; Nagashima et al, 2020 ), rectangular ( Donnelly et al, 2010 ), or elliptical ( Akiyama et al, 2021 ) shapes. Moreover, CSA may also be determined from immunohistochemical sections.…”

“…In contrast, histological studies of in vitro models have shown that myotubes are mainly in the outer ring of the constructs rather than homogeneously distributed along the cross-section ( Nakamura et al, 2017 ). Thus, up to 90% of the CSA could be constituted by non-contractile tissue ( Juhas and Bursac, 2014 ; Sakar et al, 2012 ; Akiyama et al, 2021 ; Ebrahimi et al, 2021 ), likely due to the lack of proper diffusion of nutrients and oxygen within the in vitro tissue ( van der Schaft et al, 2011 ).…”

“…Indeed, the latest technological advances in tissue engineering such as 3D bioprinting or the development of novel biomaterials and culture platforms ( Capel et al, 2019 ; Denes et al, 2019 ; Costantini et al, 2021 ; Gupta et al, 2021 ; Peper et al, 2021 ) are facilitating the development of better in vitro skeletal muscle constructs. To this end, researchers have recapitulated many native skeletal muscle features, including contraction ( Madden et al, 2015 ; Urciuolo et al, 2020 ; Akiyama et al, 2021 ; Alave Reyes-Furrer et al, 2021 ), vascularization ( van der Schaft et al, 2011 ; Bersini et al, 2018 ; Osaki et al, 2018a ; Gilbert-Honick et al, 2018 ), mechanical tension ( Okano and Matsuda, 1998 ; Vandenburgh and Karlisch, 1989 ), neuromuscular junction (NMJ) ( Afshar Bakooshli et al, 2019 ; Yoshioka et al, 2020 ; Vila et al, 2021 ), and muscle stiffness ( Urciuolo et al, 2020 ). These features, represented in Figure 1 (top), are considered key requirements for generating functional and mature skeletal muscle constructs.…”

A Stretching Force Control-Based Cyclic Loading Method for the Evaluation of Mechanical Properties of Gelation Methacrylate (GelMA) Microfibers

“…For measuring the soft microfiber, a tube-tube micromanipulator system was established to provide a nano-newton actuator and sensor to handle the microfibers. In this system, two measurement strategies are provided according to two force loading modes [ 21 , 22 , 23 , 24 ]. One mode is based on the uniform elongation rate of the microfiber.…”