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

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Title: Comparison of air-liquid interface transwell and airway organoid models for human respiratory virus infection studies
Authors: Camilla T. Ekanger, Nilima Dinesh Kumar, Rosanne W. Koutstaal, Fan Zhou, Martin Beukema, Joanna Waldock, Simon P. Jochems, Noa Mulder, Cécile A. C. M. van Els, Othmar G. Engelhardt, Nathalie Mantel, Kevin P. Buno, Karl Albert Brokstad, Agnete S. T. Engelsen, Rebecca J. Cox, Barbro N. Melgert, Anke L. W. Huckriede, Puck B. van Kasteren
Contributors: RIVM Publications Repository
Source: Front Immunol
Frontiers in Immunology, Vol 16 (2025)
Publisher Information: Frontiers Media SA, 2025.
Publication Year: 2025
Subject Terms: Influenza A virus/physiology, Cells, Immunology, Cell Culture Techniques, Epithelial Cells/virology, Cell Culture Techniques/methods, Bronchi, Respiratory Mucosa, Virus Replication, Models, Biological, Respiratory Tract Infections/virology, influenza virus, Models, Influenza, Human, Humans, Bronchi/cytology, Respiratory Tract Infections, Cells, Cultured, Cultured, Organoids/virology, Cytokines/metabolism, Human/immunology, Epithelial Cells, RC581-607, Biological, respiratory tract, Influenza, Organoids, Respiratory Mucosa/virology, complex in vitro models, Influenza A virus, mucosal models, harmonization, Cytokines, Immunologic diseases. Allergy
Description: IntroductionComplex 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.MethodsIn 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.ResultsThe 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.DiscussionIn 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.
Document Type: Article
Other literature type
ISSN: 1664-3224
DOI: 10.3389/fimmu.2025.1532144
Access URL: https://pubmed.ncbi.nlm.nih.gov/39981254
https://doaj.org/article/58f22a426f6a48649e6b3149113b6e98
https://rivm.openrepository.com/handle/10029/628397
Rights: CC BY
Accession Number: edsair.doi.dedup.....dd0c5f641c3d32777bcc40fa537b3f83
Database: OpenAIRE
Description
Abstract:IntroductionComplex 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.MethodsIn 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.ResultsThe 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.DiscussionIn 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.
ISSN:16643224
DOI:10.3389/fimmu.2025.1532144