Comparison of air-liquid interface transwell and airway organoid models for human respiratory virus infection studies

Camilla T Ekanger ^1,2,3 Nilima D Kumar ⁴ Rosanne W Koutstaal ⁵ Fan Zhou ³ Martin Beukema ⁴ Joanna Waldock ⁶ Simon P Jochems ⁷ Noa Mulder ⁸ Cecile A C M Van Els ^5,9 Othmar G Engelhardt ⁶ Nathalie Mantel ¹⁰ Kevin P Buno ¹¹ Karl Albert Brokstad ^12,13 Agnete S T Engelsen ^1,2 Rebecca J Cox ^14,3 Barbro N Melgert ^15,8 Anke L W Huckriede ⁴ Puck B Van Kasteren ^5*

• ¹ Department of Biomedicine, Faculty of Medicine, University of Bergen, Bergen, Hordaland, Norway
• ² Centre for Cancer Biomarkers, Faculty of Medicine, University of Bergen, Bergen, Hordaland, Norway
• ³ influenza Centre, Department of Clinical Science, University of Bergen, Bergen, Hordaland, Norway
• ⁴ Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, Groningen, Netherlands, Netherlands
• ⁵ Center for Infectious Disease Control (CIb), National Institute for Public Health and the Environment (Netherlands), Bilthoven, Netherlands
• ⁶ Influenza Resource Centre, Vaccines, Science Research & Innovation, Medicines and Healthcare products Regulatory Agency (United Kingdom), London, United Kingdom
• ⁷ Leiden University Center for Infectious Diseases (LU-CID), Leiden University Medical Center (LUMC), Leiden, Netherlands
• ⁸ Groningen Research Institute for Pharmacy, Department of Molecular Pharmacology, University of Groningen, Groningen, Netherlands
• ⁹ Section Immunology, Department Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, Netherlands
• ¹⁰ Sanofi, Marcy L'Etoile, France
• ¹¹ GlaxoSmithKline (Italy), Siena, Tuscany, Italy
• ¹² Influenza Centre, Department of Clinical Science, University of Bergen, Bergen, Hordaland, Norway
• ¹³ Department of Safety, Chemistry and Biomedical Laboratory Sciences, Western Norway University of Applied Sciences, Bergen, Hordaland, Norway
• ¹⁴ Department of Microbiology, Haukeland University Hospital, Bergen, Hordaland, Norway
• ¹⁵ Groningen Research Institute for Asthma and COPD, University Medical Center Groningen, Groningen, Netherlands, Netherlands

Complex in vitro respiratory models, including air-liquid interface (ALI) transwell cultures and airway organoids, have emerged as promising tools for studying human respiratory virus infections. These models address several limitations of conventional two-dimensional cell line and animal models. However, the lack of standardized protocols for the application of these models in infection studies limits the possibilities for comparing results across different research groups. Therefore, we applied a collaborative approach to harmonize several aspects of experimental methodology between different research laboratories, aiming to assess the comparability of different models of human airway epithelium in the context of respiratory viral infections. 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. The presence of club, goblet, and ciliated cells was confirmed in all models by immunofluorescent staining of the tissues. Infection kinetics were compared between the models, using a shared stock of influenza A virus as a model pathogen. We observed similar replication kinetics with a >4-log increase in virus titer across all models using a TCID50 assay. Next, we compared the antiviral host response between the models by assessing the epithelial cytokine response to virus infection using a multiplex immunoassay. 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, virus neutralization was assessed in each of the models 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 titers as quantified by TCID50. 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 in vitro respiratory models for future studies.