Purification and Culture of Airway Basal Cells

Under a protocol approved by the Weill Cornell Medical College Institutional Review Board, healthy nonsmokers were recruited for this study. The subjects were confirmed to be nonsmokers by urine levels of nicotine (<2 ng/ml) and cotinine (<5 ng/ml) with normal pulmonary function tests and chest X-ray. Following written informed consent, flexible bronchoscopy was used to collect large airway epithelial cells by brushing the epithelium [47 (link)-49 (link)]. Basal cells (BC) were subsequently purified from the total airway epithelium brushings by trypsinization of the cells and selective culturing of BC on T25 cm² plastic tissue culture flasks as previously described [4 (link),50 (link)]. The airway epithelial cells collected by brushing were pelleted by centrifugation (250 × g, 5 min) and disaggregated by resuspension in 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) for 5 min, at 37°C. Trypsinization was stopped by addition of HEPES buffered saline, (Lonza, Basel, Switzerland) supplemented with 15% fetal bovine serum (FBS; GIBCO-Invitrogen, Carlsbad, CA), and the cells were again pelleted at 250 × g, 5 min. The pellet was resuspended with 5 ml of phosphate buffered saline, pH 7.4 (PBS), at 23°C, then centrifuged at 250 × g, 5 min. Following centrifugation, the PBS was removed and the cells resuspended in 5 ml of Bronchial Epithelial Growth Media (BEGM, Lonza, CA) and 5 × 10⁵ cells plated in T25 flasks in 5 ml of BEGM and maintained in a humidified atmosphere of 5% CO₂ at 37°C. The next day, unattached cells were removed by changing the medium and thereafter, every 2 days. Following 7–8 days of culture, when the cells were 70% confluent, they were characterized by immunohistochemical staining of tryspinized cytopreps using cell type specific markers as being >99% BC (KRT5⁺, TP63⁺, CD151⁺, β-tubulin IV^-, MUC5AC^-, TFF3^-, CC10^-, chromogranin A^- and N-cadherin^-) and when put on air-liquid interface (ALI) culture, were capable of differentiating into a mucociliary epithelium [4 (link)]. To passage the cells, the primary BC were seeded at a cell density of 3000 cells/cm² in BEGM. The following day, the media was replaced with fresh BEGM and thereafter, every 2 days.