DEVELOPMENT OF HUMAN DISC-ON-A-CHIP FOR INTERVERTEBRAL DISC USING MICROFLUIDIC AND HYDROGEL SYSTEMS

Discogenic back pain is primarily caused by intervertebral disc (IVD) degeneration, driven by extracellular matrix (ECM) dysregulation, inflammation, and sensory nerve ingrowth. Studies of IVD degeneration have relied on animal models and traditional in vitro systems, which fail to replicate the cellular interactions within the IVD's microenvironment. To address, we developed a human disc-on-a-chip — miniature IVD model integrating a microfluidic gradient device and cell-encapsulated hydrogels mimicking the IVD's heterogeneous structure: nucleus pulposus (NP), annulus fibrosus (AF), and sensory neurons. We designed a disc-on-a-chip by varying inlet microchannel configurations and chamber shapes to represent NP, AF, and sensory regions. Flow rate versus fluid velocity was simulated computationally within the system. The device was fabricated using polydimethylsiloxane (PDMS) through stereolithography with a customised 3D-printed mold, plasma-treated and bonded to microelectrode array (MEA) glasses. PDMS slab was characterised for surface hydrophilicity and roughness. Hydrogels mimicking IVD ECM were fabricated by varying concentration and composition: HA/COLII for NP and COLI for AF. iPSC-derived neurons were incorporated in COLI hydrogel, peripheral to AF region. Hydrogels were characterised for mechanical properties, and cell-encapsulated hydrogels for viability, metabolic activity, and expression of phenotype markers: COL2A1 , KRT19 , ACAN , Brachyury for NP; COL1A1 , FN for AF; and peripherin, CGRP, NaV1.7, PGP9.5 for sensory neurons. Sensory activity was assessed by local field potential and calcium imaging. Disc-on-a-chip was designed with distinct NP, AF, and sensory chambers. The 3D design featured linear inlet microchannels for symmetrical flow, while curved inlets and chambers enabled flow direction modulation. Computational simulation showed pressure-dependent increases in shear stress and velocity, while curved design provided uniform flow and effective pressure dissipation. Oxygen-plasma-treated PDMS slabs successfully bonded to glass slides; prolonged plasma activation increased surface roughness and hydrophilicity. Rheological profiles showed increasing HA/COLII concentration to 3?mg/ml increased storage modulus, viscosity, and shear stress. Optimal viscosity and lower shear resistance observed at 2?mg/ml, offering more favourable environment for encapsulated cells. NP and AF cells showed high viability and metabolic activity across hydrogel groups. NP cells appeared rounded, well-distributed, with higher expression of COL2A1, KRT19, ACAN, and Brachyury, while AF cells expressed COL1A1 and FN, confirming region-specific phenotypes. We developed a disc-on-a-chip with distinct NP, AF, and sensory regions. Our findings suggest that disc-on-a-chip provides relevant model of the human IVD in vitro device system for disease modelling and drug screening.