Primary culture of human bronchial epithelial cells in air/liquid interface (ALI) is a validated in vitro model, which provides fully differentiated and pseudostratified bronchial epithelium and allows to analyze its functions in different respiratory diseases. However, the potential impact of the culture medium on the structure and the metabolism of this reconstituted epithelium is currently underestimated.

Epithelial cells were grown either in PneumaCult Ex (PC Ex) or PneumaCult Ex+ (PC Ex+) medium (StemCell, Vancouver, Canada). After the expansion phase, cells were differentiated in PneumaCult ALI medium. Analyses were performed during the expansion phase, and at day 0 (D0), D7, D14, D21 and D28 of the differentiation process. Pictures of the cell morphology were taken, immunostaining were carried out on cytocentrifuged cells, and culture supernatants were stored for subsequent analyses. At D28 of differentiation, bronchial epithelium was challenged with Poly I:C or TNF-α. The cytokine secretion in culture media or apical lavages, and the expression of mRNA and intra-cellular protein were assessed.

PC Ex+ medium provided a higher cell yield than PC Ex. Morphology was found to be different, as PC Ex+-cultured cells formed clusters. At early and differentiated stage, cells cultured in PC Ex medium expressed specific epithelial and mesenchymal markers (pan-cytokeratin and vimentin, respectively), while cells in PC Ex+ mainly expressed the epithelial marker.

Our preliminary results showed differences in terms of cellular yield and epithelial-mesenchymal cell features, depending on the culture medium used for cellular expansion. The influence of the expansion medium on cell subtypes during the differentiation steps is under investigation. In the near future, we will compare the release of cell mediators at steady state and after stimulation to better understand the effect of culture medium on the functional responses of epithelial cells.