Isolation of Human Pulmonary Epithelial Cells    

1. Pulmonary epithelial cells from human lung tissue were isolated.
2. Lungs were perfused with 10 ml of primary cell medium (supplemented with 1% antibiotics and 2 mM L-glutamine) through the pulmonary artery until they were cleared of blood.
3. Bronchoalveolar lavages were performed using 5 × 1 ml of PBS, 1 mM EDTA to remove alveolar leukocytes.
4. One ml of primary cell isolation solution was instilled into the lungs through the trachea.
5. Lungs were then removed and placed in a sterile tube containing 2 ml of primary cell medium (supplemented with 1% antibiotics and 2 mM L-glutamine) and incubated at 4 °C overnight for enzymatic digestion to occur.
6. Lung tissues were further teased apart in primary cell medium, 10% FCS in a dish.
7. The cell isolate was passed through a 100-μm filter, and pulmonary epithelial cells were purified as follows.
8. Collected cells were counted and incubated for 15 min at 4 °C at the appropriate ratios.
9. Cells were then washed and diluted in primary cell medium, 0.5% FCS.
10. Pulmonary epithelial cells thus obtained were plated on 6-well plate (4 × 10⁵ cells/well) in primary cell medium, 10% FCS.