MULTI-STIMULUS FUSION PLATFORM FOR PROMOTING C2C12 ALIGNMENT AND MYOTUBE FORMATION

The development, maturation, and functionalization of skeletal muscles require the synergistic effects of mechanical and electrical stimulation.Here we use an organ-on-a-chip to integrate two different stimulation sources.Uniaxial pre-stretching can reduce the heterogeneity of C2C12 myoblast arrangement; followed by electrical pulse stimulation to promote functional differentiation of myotubes.This platform holds great promise in realize the functions of cells and tissues by regulating the multi-source microenvironment. INTRODUCTIONThe extracellular matrix (ECM) has a decisive influence on the development and functionalization of cells and tissues 1,2 .The process of skletal muscle fusion into multinucleated myotubes requires multi-step regulation of mechanical and electrical stimulation.Research on skeletal muscle tissue engineering using organ-on-a-chip has made remarkable progress 3 .However, there are still several problems to be solved.Uniform uniaxial mechanical stretching is difficult to achieve, and patternable, highly biocompatible electrodes are difficult to manufactured.Therefore, it is necessary to integrate multi-source stimulation (mechanical stretch and electrical pulse) on organ-on-a-chip to regulate the microenvironment of myoblast arrangement and myotube generation, so as to study the impact of multi-source stimulation on the behavior of skeletal muscle cells. MATERIAL & METHODSThe organ-on-a-chip designed in Figure 2 completes the integration of two stimulus sources.Cells are cultured by perfusing cell suspension and media into the cell culture chamber.The quilibrium liquid chamber can balance the longitudinal movement of the stretched film above, thereby obtaining uniaxial stretching of the film.Symmetry vacuum chambers on both sides of the long axis of the culture chamber drives the cells on the PDMS membrane to stretch.PDMS-CNT electrodes are provided on both sides of the short axis of the culture chamber.During the differentiation stage, electrical pulses are applied to the myotubes to promote myotube functionalization. RESULT & DISCUSSIONThis work uses organ-on-a-chip to integrate two different stimulation sources to study the entire process of myoblast alignment and differentiation, giving full play to the advantages of organ-on-a-chip in providing multi-source stimulation during cell and tissue culture.Uniaxial stretching of the film through the equilibrium liquid chamber is an innovative point in this study.Replacing different "balancing liquids" in the same structure can be compatible with different stretching magnitudes.In addition, the PDMS-CNT electrode is easy patterned molding and has a good biocompatibility, which has great advantages in PDMS-based chips.Figure 3 shows the effect of uniaxial cyclic stretching on cell number and arrangement.After 3 days of uniaxial cyclic stretching, the number of myoblasts was 1/3 of that in the control group.The quantitative control of the cells made the myoblasts a monolayer structure, which was conducive to the generation of monolayer myotubes.At the same time, the oriention arrangement of cells which concentrated in 9030also has great advantages.Arrangement in the same direction is very beneficial to cell differentiation.Further experiments and characterization of the differentiation function of electrical pulse stimulation are needed in the future.