PriCells: Isolation, culture, and purification of Human Pulmonary Microvascular Endothelial Cells (HPMECs)

1. HPMECs were isolated from lungs resected from tissue of normal human or adult patients with malignant tumors.
2. Normal lung tissue or adult patients with malignant tumors from the subpleural region (1g) was removed and minced.
3. The tissue fragments were then incubated under gentle agitation with dispase (0.4%) of PriCells Isolation Kit at 4°C for 12 hours.
4. Cell debris and erythrocytes were removed by filtration through a 100-m nylon mesh strainer (Becton Dickinson Plasticware).
5. The tissue fragments were digested with PriCells Isolation Kit for 30 minutes at 37° C after which the suspension was homogenized by pipetting.
6. The homogenate was filtered first through a 100-m mesh strainer and subsequently through a 40-m mesh strainer and pelleted by centrifugation (173g for 10 minutes).
7. The cell pellets were resuspended in Pricells Culture System and c*ted on 6-well plates pre-coated with gelatin (0.2%) for several days until subconfluent.
8. Purification: Endothelial cells were isolated using magnetic beads (Dynal) according to the instructions of the manufacturer.
9. The medium was replaced every 2 to 3 days, and the subcultures were obtained by trypsin/EDTA treatment of confluent monolayers at a splitting ratio of 1:3.
10. Identification: Cells were identified with PriCells Identification System.