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

Diseases or injuries that impact neuromuscular tissues severely impact human health, motivating the development of in vitro models of the motor control system. Current platforms either have limited reproducibility or require complex fabrication protocols that preclude high-resolution imaging and scalable functional analysis. We have developed a one-step method for fabricating an all-hydrogel Limb-on-a-Chip that addresses key challenges of current systems by enabling reproducible and scalable manufacturing of neuromuscular tissues compartmentalized into "spinal cord" and "limb" chambers. Co-cultures of motor neurons and skeletal muscles within this platform enable longitudinal tracking of neuromuscular junction formation and visualizing changes in muscle contraction in response to motor neuron stimulation. We demonstrate tissue-wide recordings of muscle force and single cell-resolution measurements of muscle calcium activity. Our accessible method for fabricating reproducible in vitro neuromuscular models compatible with high-resolution imaging and functional readouts provides a powerful tool for investigating the neuromuscular interface in health and disease.