Cell Culture
Human bronchial epithelial BEAS-2B cells were cultured in RPMI growth medium, supplemented with 10% FCS, 1% penicillin/streptomycin at 37°C in a humidified incubator at 5% CO2. Human primary bronchial epithelial cell cultures (primary HBE) were bought from Lonza (Visp, Switzerland) or isolated from biopsies from individuals treated by lobectomy because of non-small-cell lung carcinoma at the Thoraxklinik Heidelberg. Ethics approval (S-381/2014) was obtained from the regional Ethics Committee at the University of Heidelberg, and all study participants provided a written informed consent. Two methods of bronchial epithelial cells isolation were used (65, 66): Briefly, the obtained biopsies were washed with cold EBSS and cleaned from any additional connective tissue and mucus. The segments were then cut open and incubated on a shaker at 4°C overnight in 9 ml EBSS including 1 ml digestion solution (DS, 10x: 0.01% DNase I, 1% Protease XIV in sterile PBS). On the next day, 1.1 ml FCS was added to the solution to terminate digestion. The epithelium was scraped into the digestion medium using a sterile scalpel. The biopsies were additionally washed with EBSS. All epithelial cells from scraping were collected by spinning at 4°C at 500 × g for 5 min. Cell pellets were resuspended in 10 ml of warm BEGM (Lonza) and cells were then seeded into collagen-coated (3 mg/ml PureCol, Cellsystems, 1:75 in ddH2O) prewarmed 10 cm culture dishes.
Additionally, isolation using an outgrowth method was performed: Bronchial segments were cut into 2–3 mm3 pieces and placed into collagen-coated 6-well-plates. These pieces served as a source of primary cells and grown out epithelial cells were transferred into collagen-coated 10 cm dishes as soon as they reached 70% confluence. Primary cell cultures were maintained at 37°C in a humidified incubator at 5% CO2. Culture medium (BEBM + Supplements) was changed every 2–3 days. Cells were passaged when reaching 70–90% confluency. After 2 washes with PBS, they were trypsinized with 2 ml of 0.05% trypsin/EDTA and incubated 5–10 min at 37°C. Cells were then rinsed twice with PBS, harvested and pooled into a tube containing soybean trypsin inhibitor (STI, 1 mg/ml) on ice. After spinning at 500 × g for 5 min, the pellets were resuspended in 10 ml BEGM and cell suspension was split into two new collagen-coated 10 cm dishes.