Tracheas were obtained from one-day-old SPF chicks and rinsed in DMEM medium (Invitrogen) under sterile conditions. After the removal of surrounding adipose and muscular tissues, tracheas were digested with dispase I solution (2.5 U/mL dispase I) (Roche, Nutley, NJ, USA) for 2 h at 37°C to disrupt the basal membrane. Forceps were used to force the outflow of detached cell sheets of tracheal epithelium from the tracheal lumen. The epithelial cells were harvested and further digested with collagenase I (1 mg/mL, Roche) for 5 min at 37°C. The cells were gently pipetted, and the tissues were homogenized into small cell clumps. FBS was added to the digesting solution to stop the reaction. The cells were centrifuged at 1000 rpm for 5 min to remove residual digesting enzymes. The cell pellets were resuspended in ATE medium, containing 10% FBS (Invitrogen), 10% chicken embryo extract (CEE) (US Biological, Swampscott, MA, USA; or self-made [27] ), 1% glutamine (Invitrogen), 0.1 mM b-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 1% nonessential amino acids (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) in DMEM-F12 medium (Invitrogen). Cells were seeded on 2% matrigel-or 20 lg/mL fibronectin-coated 24-well or 96-well plates (Corning Inc., Corning, NY, USA). When the cells reached confluence, the cultured cells were passaged by digestion with both 0.01% (w/vol) protease (type XIV, Sigma-Aldrich) and 2.5 U/mL dispase I for 5 min at 37°C.