Developing analysis algorithm for engineered skeletal muscle contractile function based on a microphysiological system

Autonomous contractility is a key functional characteristic of skeletal muscle and an important indicator of engineered muscle maturation. Conventional contact-based force measurements are limited by mechanical interference, restricted spatial resolution, and poor compatibility with high-throughput microphysiological systems. Here, we present a non-contact optical–mechanical framework for quantitative evaluation of contractile dynamics in three-dimensional engineered skeletal muscle. C2C12-derived muscle bundles were generated in fibrin–Matrigel hydrogels and cultured within a customized PDMS microdevice incorporating dual elastic cantilevers and integrated electrodes for controlled electrical stimulation. High-resolution video microscopy combined with optical flow tracking was used to quantify cantilever deformation, which was subsequently converted into contractile force using cantilever beam theory. This approach enabled the extraction of multiple functional parameters, including contraction amplitude, frequency, velocity, and force–time characteristics. The engineered tissues formed aligned, striated myofibers and exhibited rhythmic contractions synchronized with stimulation. Quantitative analysis demonstrated a strong correlation between separately recorded optical deformation and calcium transient signals (r = 0.88 ± 0.03, n = 5 independent constructs, p < 0.01). Reproducible force outputs and frequency-dependent changes in deformation amplitude and contraction velocity were observed across stimulation frequencies from 1 to 3 Hz. Estimated cantilever forces were consistently higher on the right than on the left, suggesting a possible contribution from systematic device- or measurement-related factors. Overall, this framework provides a non-invasive and quantitative approach for functional assessment of engineered skeletal muscle and may facilitate applications in disease modeling, drug evaluation, and microphysiological system-based research.