Fig. 1.

Primary culture of mouse middle ear epithelial cells. (A) Timeline for culture of mMECs. Bullae were dissected, treated with pronase for dissociation of the middle ear epithelial cells and fibroblasts were excluded from culture by differential adherence to plastic. Epithelial cells were grown in submerged culture until confluence, before ALI was induced. Samples for transcriptional and proteomic analysis were collected at regular time points. (B-I) Phase-contrast images showing cells in culture under 10× magnification. Under the proliferative submerged conditions (SUB), a small number of cells attached to form epithelial islands 3 days after seeding (B). The cells proliferated faster from day (D)5 (C) through day 7 (D) and formed a confluent monolayer at day 9. This was termed ALI day 0 (E). Morphology of cells changed from ALI day 3 (F) and clusters of compactly arranged cells started forming at ALI day 7 (G). (H) ALI day 14 cultures were composed of flat polygonal and compactly clustered pseudostratified cells with active cilia. White arrows mark elevated ciliated cells and asterisks mark flatter polygonal cells. (I) Fibroblasts cultured on plastic plates through differential adhesion method. Scale bars: 200 μm.