Spencer, S. M. | Search bioRxiv

bioRxiv2025

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.

microbiology↗

bioRxiv2023

Modeling Cystic Fibrosis Chronic Infection Using Engineered Mucus-like Hydrogels

The airway mucus of patients with cystic fibrosis has altered properties which create a microenvironment primed for chronic infections that are difficult to treat. These complex polymicrobial airway infections and corresponding mammalian-microbe interactions are challenging to model in vitro. Here, we report the development of mucus-like hydrogels with varied compositions and viscoelastic properties reflecting differences between healthy and cystic fibrosis airway mucus. Models of cystic fibrosis and healthy airway microenvironments were created by combining the hydrogels with relevant pathogens, human bronchial epithelial cells, and an antibiotic. Notably, pathogen antibiotic resistance was not solely dependent on the altered properties of the mucus-like hydrogels but was also influenced by culture conditions including microbe species, monomicrobial or polymicrobial culture, and the presence of epithelial cells. Additionally, the cystic fibrosis airway model showed the ability to mimic features characteristic of chronic cystic fibrosis airway infections including sustained polymicrobial growth and increased antibiotic tolerance.

bioengineering↗