Evolution of NPTr cells in ALI conditions over the twenty-two days of culture. A-D: morphological evolution, representative images of two independent experiments. E-J: immunostaining and scanning electron microscopy assessment. NPTr cells were cultured in ALI conditions over twenty-two days and the aspect of the cellular layer was assessed by immunofluorescence and scanning electron microscopy. E: After reaching confluence, NPTr cells were cultured in ALI conditions for one week. F: After twenty-two days in ALI conditions, NPTr cells showed a thick cellular layer with the presence of numerous mucin positive cells and a delineated border of tubulin positive cells. G and H: immunostaining of the bronchial epithelium. Tissue was stained with an antibody recognizing mucin 5 AC (green, G) or with an antibody recognizing β-tubulin (red, H). I and J: Using scanning electron microscopy, a cellular layer showing a more complex topography and covered by a thick mucus layer was observed.