Limb-on-a-Chip: An All-Hydrogel Platform for Scalable and Reproducible Engineering of Neuromuscular Tissues

Diseases or injuries that impact neuromuscular tissues have a severe negative impact on human health, mobility, and quality-of-life, motivating the development of tissue engineered in vitro models of the motor control system. Current neuromuscular organoids and organ-on-a-chip platforms either rely on stochastic self-assembly that limits reproducibility or require complex microfabrication processes that preclude high-resolution imaging and scalable functional analysis. We have developed a Limb-on-a-Chip platform that addresses key challenges of current model systems by enabling reproducible and scalable manufacturing of neuromuscular tissues compartmentalized into "spinal cord" and "limb" chambers, while promoting biochemical crosstalk between cell types. Our fabrication method leverages 3D printed molds to perform 1-step micropatterning of an all-hydrogel chip containing precise features to guide muscle fiber alignment and motor neuron axonal outgrowth. We demonstrate the ability to co-culture motor neurons and skeletal muscles within this hydrogel platform, enabling tissue-wide readouts of muscle force as well as single cell-resolution measurements of muscle fiber calcium activity. Our accessible method for fabricating reproducible in vitro neuromuscular models that are compatible with high-resolution imaging and functional readouts provides a powerful new tool for investigating the neuromuscular interface in health and disease.