Developing a Functional in Vitro Model of the Neuromuscular Interface

Abstract

The neuromuscular system is responsible for the coordination of movement throughout the body, and while research has revealed many of the mechanisms involved in the function of the neuromuscular system, there are still many gaps in our understanding of how all of the components of the system work and how they are affected by environmental factors and disease. This work focuses on developing methods and an in vitro model for studying a subsystem of the neuromuscular system known as the neuromuscular junction (NMJ), which is the connection between skeletal muscle and motor neurons and is relevant in many neuromuscular degenerative diseases. This work identifies that current in vitro NMJ models are cohesively lacking the ability to support long-term, functionally contractile muscle tissue while providing compartmentalization and clear optical access for live imaging of muscle and motor neuron co-cultures. This work therefore presents STAMP, a microgroove patterning method for creating aligned, more physiologically relevant, functional, and optically accessible skeletal muscle tissue cultures on top of fibrin hydrogels. Through investigating a series of different sizing parameters, STAMP is shown to effectively align mouse and human skeletal muscle monolayers in vitro and influence the direction of muscle contraction under electrical and optogenetic stimulation while preserving skeletal muscle tissue integrity and viability. The STAMP approach provides a way to mold hydrogels and the morphology of muscle tissue and will be beneficial for addressing the need for compliant and optically clear substrates in modeling the neuromuscular junction.