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.