A 3D-printed multi-well lung-on-chip platform with bioprinted stromal compartment for upper airway tissue culture and antiviral drug evaluation.

Physiologically relevant lung-on-chip (LoC) systems remain constrained by polydimethylsiloxane (PDMS) limitations including small-molecule adsorption and mechanical deformation of device microchannels under perfusion, single-channel architectures limiting parallel tissue culture, and absence of organized stromal and immune compartments. Existing platforms predominantly model the distal airway, leaving the upper airway, the primary site of respiratory viral entry, insufficiently addressed as an in vitro tissue model for preclinical drug evaluation. We report a six-well, 3D-printed LoC platform fabricated using a photopolymer resin that addresses these limitations. Computational fluid dynamics (CFD)-guided design ensured uniform perfusion across all six wells, enabling parallel tissue culture within a single device. The photopolymer architecture eliminated mechanical deformation of the device microchannels under prolonged perfusion and demonstrated significantly reduced small-molecule adsorption relative to PDMS, improving quantitative reliability for drug evaluation workflows. Human tracheobronchial epithelial cells differentiated at air-liquid interface expressed MUC5AC and cytokeratin-14, formed continuous ZO-1 tight junctions, and achieved a transepithelial electrical resistance of ~800 Ω·cm2, consistent with physiological barrier integrity. A GelMA-based stromal compartment, fabricated by extrusion bioprinting of lung fibroblast- and THP-1 monocyte-laden bioink, established a layered epithelial-stromal-immune architecture. Functional validation demonstrated IL-6 and IL-8 elevation following SARS-CoV-2 spike S1 stimulation, IFN-γ-driven modulation of apical ACE2 expression, and coordinated TNF-α, IL-6, and IL-8 release during Influenza A infection, all suppressed by oseltamivir treatment. This platform integrates CFD-optimized multi-well device fabrication, extrusion bioprinting of stromal constructs, and photopolymer resin-based microfluidic manufacturing into a reproducible, human-relevant upper airway model for respiratory infection research and preclinical antiviral drug evaluation.