Optimizing respiratory virus replication in human bronchial/tracheal epithelial air–liquid interface cultures through practical quality indicators and key characteristics

The human bronchial/tracheal epithelial air–liquid interface (HBTEC-ALI) culture system is widely used for isolating and propagating respiratory viruses; however, variability in differentiation status complicates quality control and may influence viral replication efficiency. Practical criteria for selecting optimally differentiated cultures remain limited. In this study, we classified HBTEC-ALI cultures into three morphological categories – Thick, Intermediate and Thin – based on cell size, ciliary activity and mucus production. These categories were associated with epithelial thickness and expression of the basal cell marker KRT5 . Thin-type cultures exhibited significantly reduced transepithelial electrical resistance and diminished apical glycan staining, suggesting compromised barrier integrity and altered apical glycan composition. We next evaluated replication of multiple respiratory viruses. Most viruses tended to replicate more efficiently in Thick- and Intermediate-type cultures, whereas adenovirus replication was enhanced in Thin-type cultures. Bocavirus replication did not differ between types. Our findings suggest that morphological assessment of HBTEC-ALI cultures is associated with functional barrier status and virus-specific replication efficiency. In addition, the association between KRT5 expression and morphological phenotypes suggests that KRT5 expression may reflect differences in epithelial characteristics among HBTEC-ALI cultures. These results may provide a practical framework for selecting HBTEC-ALI cultures and may help improve the reproducibility and efficiency of respiratory virus isolation and propagation.