A 3D Human Neuron‐on‐Chip Platform to Monitor Neuronal Injury Responses

Traumatic brain injury (TBI) is a major cause of neurological dysfunction and long-term neurodegeneration, yet the intrinsic neuronal contributions to TBI pathophysiology remain incompletely defined. Here, we present a novel Neuron-on-Chip microfluidic device and weight-drop impactor platform that can be used to mechanically injure mature human prefrontal cortex neurons (hPFCs) embedded in three-dimensional (3D) hydrogels, enabling the study of injury responses in pure neuronal cultures. We assessed real-time calcium dynamics across 13 metrics of single-cell and network activity, revealing a biphasic injury response: an early phase (0.5-24 h) characterized by excitotoxicity, hyper-synchronized bursting, and network collapse; and a late phase (8 d) marked by sustained depolarization and structural remodeling. Secretome profiling revealed progressive elevations in extracellular pT181 and total Tau from days 1 to 5 post-injury. Cytokine analysis identified early (24 h) elevations in IP-10, IL-10, IFNα2, and NCAM, and late increases (8 d) in CXCL9 and MPO, linking neuronal activity changes to stage-specific inflammatory signaling. Immunocytochemistry and immunoblotting confirmed temporally ordered upregulation of calpain-1 and active caspase-3 (days 1-3), phosphorylated Tau (AT8+, days 5-8), and neurofibrillary tangle-like Tau aggregates (NFT+, day 8). These findings establish our platform as a scalable microphysiological model for probing the dynamic cellular and molecular sequelae of neuronal response to injury, offering insights into neurodegeneration and opportunities for therapeutic discovery.