Bottom‐Up Engineering of a Human Neuromuscular System for Modeling Activity‐Induced Remodeling, Metabolic Stress, and Endothelial‐Modulated Excitability

In vitro models of the human neuromuscular system recapitulate key features of neuromuscular connectivity and are increasingly used to study disease mechanisms. However, activity-dependent adaptation and the contribution of endothelial cells (ECs) remain incompletely represented in vitro. Here, we establish a bottom-up, microchip-based method that supports motor innervation of three-dimensional (3D) human muscle and permits local or bath application of defined chemical stimuli. The same architecture also supports endothelial ingrowth, producing myobundles with concurrent neural and endothelial integration. Repeated local L-glutamate stimulation induced structural, metabolic, and transcriptional changes associated with activity-dependent muscle adaptation, whereas high-glucose exposure produced a distinct, largely opposing response. Addition of ECs further altered calcium dynamics in motor neurons (MNs) and innervated muscle fibers. This modular method enables controlled investigation of responses to neural, metabolic, and endothelial cues in engineered human neuromuscular system.