A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial cultures at air-liquid interface (HAE) are a physiologically relevant in vitro model of this heterogeneous tissue, enabling numerous studies of airway disease[1][1]–[7][2]. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining capacity for differentiation to HAE[5][3]. However, gene expression and innate immune function in HAE derived from BCi-NS1.1 versus primary cells have not been fully characterized. Here, combining single cell RNA-Seq (scRNA-Seq), immunohistochemistry, and functional experimentation, we confirm at high resolution that BCi-NS1.1 and primary HAE cultures are largely similar in morphology, cell type composition, and overall transcriptional patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1 HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus . Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-7 [3]: #ref-5