P036 A comparative study of cell culture inserts for in vitro modeling of the cystic fibrosis airway epithelium

Objectives: To generate physiologically relevant models to study the human airways, researchers are relying on cell cultures grown as organoids in hydrogel or Air-liquid interface (ALI) cultures in transwells. A consequence of the coronavirus (COVID-19) pandemic and the current war in Ukraine, have been a global shortage on lab consumables including Corning Transwells, typically used for airway ALI cultures. One way to overcome this supply issue, is to use alternative cell culture inserts. In this study, we compared ALI cultures grown on available commercial inserts, to ensure exchangeability and reproducibility across brands. Methods: Airway epithelial basal cells were seeded on the inserts and differentiation was monitored over a 4 week period. We used an immortalized cell line and primary cells from CF and non-CF airways. After 4 weeks in ALI culture, we measured cell layer thickness, epithelial integrity, and cellular composition. The cultures were additionally tested as infection models, measuring bacterial penetration, cytotoxicity, and cytokine secretion upon 14 hours of co-culture with bacteria specifically prevalent in CF airways. Results: We found significant morphological differences in the ALI cultures grown in different inserts, this included variation in both cell layer thickness and differentiation status. Several tested inserts supported no or only limited differentiation of primary cells. Upon bacterial infection, we detected differences in bacterial localization and penetration, cytotoxicity and levels off cytokine secretion. Conclusions: Comparing exchangeability of culture systems are essential to ensure reproducibility in experiments before changing components. Choice of culture system may severely influence both disease modeling and host-microbe interaction studies. Several inserts did not support differentiation of primary CF cells, this limits our ability to model CF airways in vitro, and may have severe negative impact on future CF research.