Tracheas were obtained from one-day-old SPF chicks (Animal Health Research Institute, Tansui, Taipei, Taiwan) and rinsed in a DMEM medium (Invitrogen, Carlsbad, CA, USA) under a sterile condition. The procedure for removing epithelial sheets from the tracheas and the detailed culture conditions for the CTE cells have been described previously [21] . Briefly, tracheas were digested with dispase I solution (2.5 U/ml dispase I, Roche) for 2 h at 37uC. The detached cell sheets of tracheal epithelium from the tracheal lumen were harvested and further digested with collagenase I (1 mg/ml, Roche) for 5 min at 37uC. The disrupted tracheal epithelial sheets were gently pippeted and homogenized into small cell clumps. The cell pellets were resuspended in a CTE medium [21] and were seeded on 2% matrigel-coated 24-well plates (Corning). The cells were cultured at 37uC with 5% CO 2 for 3 days. The animal use protocol in this study had been reviewed and approved by Institutional Animal Care and Use Committee in National Chung Hsing University. The approval number is 99-09.