Validation of in vitro models for smoke exposure of primary human bronchial epithelial cells

Abstract

The bronchial epithelium is constantly challenged by inhalative insults including cigarette smoke (CS), a key risk factor for chronic lung diseases. Various CS exposure models for bronchial epithelial cells in vitro have been described, but often with little to no validation of efficacy or physiological relevance. Here, six different in vitro CS exposure models of primary human bronchial epithelial cells (phBECs) were validated using expression analysis of 10 genes consistently upregulated in healthy smokers, and quantification of cell-delivered dose. CS exposure of phBECs was varied in terms of bronchial epithelial cell type composition, type and duration of the exposure, dose, and application route (whole CS, CS extract). Gene expression was assessed by quantitative Real-Time PCR (qPCR) and immunoblotting while cell type-specific expression of smoke-induced genes was analyzed by immunofluorescent analysis. Three surprisingly dissimilar exposure-cell models, namely acute treatment of submerged basal phBECs with CS extract, chronic treatment of differentiating phBECs with CS extract, and whole CS exposure of differentiated phBECs, resulted in significant upregulation of 6 out of 10 genes. Acute apical or basolateral exposure of differentiated phBECs with CS extract was much less effective despite of similar doses of CS used. Overall, our findings provide important guidelines for the design of human cigarette smoke-induced in vitro models and thus encourage their use as prescreening tools prior to the consideration of animal experiments.