Stable and assayable polymicrobial human airway model reveals complex interactions between Pseudomonas aeruginosa and lung commensals

Polymicrobial-host crosstalk shapes airway barrier integrity and inflammation, yet standard in vitro systems rarely sustain interaction-dependent phenotypes. We engineered an aqueous two-phase system (ATPS) confined bronchial co-culture that stabilized day-scale assays while maintaining epithelial function. A 16HBE14o-/HUVEC cell insert model was challenged with Pseudomonas aeruginosa PA01, Streptococcus pneumoniae D39, and the commensals Rothia mucilaginosa and Lactobacillus casei in mono- and polymicrobial combinations. We evaluated epithelium permeability to FITC-dextran, cell junction integrity, bacterial-induced cytotoxicity, IL-6 and IL-8 release and bacterial viability. ATPS preserved a workable assay window and bacterial confinement over 24h. PA01 disrupted barrier integrity, and commensals mitigated this pathogenic effect, whereas PA01 co-cultured with S. pneumoniae exhibited synergistic damage effects on the lung epithelium. Junctional imaging corroborated functional readouts, and cytotoxicity remained low across conditions. Cytokine shifts were condition-specific but modest, demonstrating compatibility for soluble mediator profiling. We generated a human airway-microbiome model in which ATPS confinement enabled modelling of dynamic lung-microbiome interactions, reproducing known P. aeruginosa pathogenic effects and commensal protection of the epithelium.