For the isolation of tracheal epithelial cells, the intact cell sheet of the tracheal epithelium was first isolated from dispase-treated tracheas (Fig. 1A) . The epithelial membrane sheet was further mechanically disrupted into small pieces by pipetting. Floating unattached ATE cells rapidly underwent cell death or growth arrest after a one-day culture. The optimal cell matrix for ATE cells was evaluated; both 2% matrigel or 20 lg/mL fibronectin efficiently promoted cell attachment, but gelatin, collagen I, collagen IV and laminin were less effective (data not shown). The primary culture system for mammalian tracheal epithelial cells is well-established. To culture the ATE cells, specialized commercial media for the tracheal epithelia of humans and rats were first tested (described in the Materials and methods section). None of them supported cell growth or long-term viability of ATE cells. Adjustment of the concentration of retinoic acid, a differentiating factor for tracheal epithelial cells [10] , did not affect attachment or cell growth of ATE cells. Increasing FBS from 10% to 20% in the culture medium modestly enhanced the viability of the cells (data not shown). We speculated that unidentified chicken nutrients essential for the promotion of ATE cell growth were absent in the culture medium. CEE, an essential supplement for the culture of mouse neural crest stem cells [27] , was added to DMEM-F12 medium with 10% FBS. We found that CEE significantly improved cell growth and reduced cell death of the primary ATE cells (Figs. 1B and 1C) . The cell number of initial harvest from one trachea on day 1 is about 2 · 10 5 cells. With this optimized culture medium, isolated cells displayed increased MTT activity, and the celldoubling time was 46 h during the first 6 days of culture (Fig. 1D) . The ATE cells could sustain proliferative activity for 3-5 passages. These results show that the established culture system provides sufficient nutrients and proper 