Spatially controlled polymicrobial human airway model recapitulates complex interactions between Pseudomonas aeruginosa and lung commensals.

Airway barrier dysfunction is a hallmark of chronic lung diseases including cystic fibrosis, asthma, and chronic obstructive pulmonary disease, and is often shaped by polymicrobial rather than single-species interactions with the host tissue. However, extended host-microbe co-culture with rapid-colonizing airway pathogens such as Pseudomonas aeruginosa is difficult to sustain in accessible in vitro systems, limiting mechanistic study of polymicrobial barrier and inflammatory dynamics. Here we developed and validated an aqueous two-phase system (ATPS) that spatially confines bacterial communities over a human bronchial epithelial-endothelial transwell co-culture, extending the stable co-culture window to 24 h while preserving assayable barrier and cytokine readouts. Using a validation-by-recapitulation approach, the platform reproduced established in vivo phenomena: P. aeruginosa-driven barrier disruption, attenuation of pathogenic effects by the commensals R. mucilaginosa and L. casei with preserved junctional architecture, and S. pneumoniae exacerbation of barrier permeability accompanied by elevated IL-8 despite apparent junctional preservation. This accessible ATPS-based biomaterials platform requires no microfluidic or iPSC-derived components and provides a tractable foundation for mechanistic studies of polymicrobial interactions at the airway epithelial barrier.