INTRODUCTION: Complex METHODS: In this study, we compared three different models of human respiratory epithelium: a primary human bronchial epithelial cell-derived ALI transwell model, and two airway organoid models established from human airway- and lung-derived adult stem cells. We first assessed the presence of various differentiated cell types using immunofluorescence microscopy. Using a shared stock of influenza A virus, we then assessed viral growth kinetics, epithelial cytokine responses, and serum-mediated inhibition of infection. RESULTS: The presence of club, goblet, and ciliated cells was confirmed in all models. We observed similar viral replication kinetics with a >
4-log increase in virus titre across all models using a TCID50 assay. Following infection, a reproducible antiviral cytokine response, including a consistent increase in CXCL10, IL-6, IFN-λ1, IFN-λ2/3, and IFN-β, was detected across all models. Finally, neutralization was assessed by pre-incubation of virus with human serum. Reduced viral replication was observed across all models, resulting in a 3- to 6-log decrease in virus titres as quantified by TCID50. DISCUSSION: In conclusion, all three models produced consistent results regardless of the varying cell sources, culturing approaches, and infection methods. Our collaborative efforts to harmonize infection experiments and compare ALI transwell and airway organoid models described here aid in advancing our understanding and improving the standardization of these complex