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

Tang, R.; Latchoumane, C.-F.; Chopra, A.; Sarkar, M. M.; Kim, C.; Gonsalves, N.; Wu, H.-F.; Mhatre-Winters, I.; Mishra, A.; Zeltner, N.; Richardson, J. R.; Karumbaiah, L.

doi:10.1101/2025.08.06.667201

bioRxiv · 10.1101/2025.08.06.667201

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

Tang, R.·Latchoumane, C.-F.·Chopra, A.·Sarkar, M. M.·Kim, C.·Gonsalves, N.·Wu, H.-F.·Mhatre-Winters, I.·Mishra, A.·Zeltner, N.·Richardson, J. R.·Karumbaiah, L.

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Abstract

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 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 hr) characterized by excitotoxicity, hyper-synchronized bursting, and network collapse; and a late phase (8 d) marked by sustained depolarization and structural remodeling. Secretome profiling uncovered progressive elevations in extracellular pT181 and total Tau from days 1 to 5 post-injury. Cytokine analyses identified early (24 hr) elevations in IP-10, IL-10, IFN2, 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.

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Tang, R., Latchoumane, C.-F., Chopra, A., Sarkar, M. M., Kim, C., Gonsalves, N., Wu, H.-F., Mhatre-Winters, I., Mishra, A., Zeltner, N., Richardson, J. R., Karumbaiah, L.. 2025-08-09. A 3D Human Neuron-on-Chip Platform to Monitor Neuronal Injury Responses. https://doi.org/10.1101/2025.08.06.667201

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