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When cultured at the air-liquid interface (ALI), primary airway epithelial cells and some epithelial cell lines such as Calu-3 can polarize and form a pseudo-stratified epithelium containing progenitor providing basal cells, and columnar mucus-secreting goblet cells and ciliated cells. Bronchiolar epithelial differentiated models also contain cubic-shaped club cells. The differentiation process relies on environmental cues supplied by the presence of an ALI, the attachment of cells on a basement membrane extract (BME) such as collagen, tight cellular junctions, and the composition of the culture media. Cells first need to be expanded submerged in growth media on a BME-coated permeable support to form a monolayer with tight cellular junctions. Then, the growth media is removed and differentiation media is added only to the basolateral compartment of the permeable support system. The cells are maintained through regular media changes using differentiation media for 3-4 weeks. Within 2-3 weeks of the differentiation period, beating cilia can be observed and goblet cells begin secreting mucus. Some protocols recommend rinsing the apical chamber with buffer such as Dulbecco’s Phosphate-Buffered Saline (PBS) to prevent mucus from accumulating on the apical side of the cells.