Isolation and growth of pulmonary artery adventitial fibroblasts 

1. Adventitia from the main pulmonary artery was harvested neonatal calves.
2. Tissue was collected, carefully dissected free of blood vessels and fat under a dissecting microscope, and then cut into small pieces.
3. Adventitial tissue was isolated, carefully dissected free of blood vessels and fat under a dissecting microscope, and cut into small pieces.
4. The tissue pieces were incubated with Ca²⁺ -free HBSS for 30 min at 37°C and then with HBSS containing Ca²⁺, elastase, collagenase, albumin, and soybean trypsin inhibitor for 90 min at 37°C on a rotator.
5. The tissue was gently triturated with a sterile Pasteur pipette after each 30 min of incubation.
6. The dispersed cells were passed through a 100 μm nylon cell strainer to remove any undigested tissue pieces, diluted in Primary cell media containing 10% FBS to inactivate the enzymes, and centrifuged at 900 rpm for 10 min.
7. Using a light microscope and hemocytometer, we counted the cells and serially diluted this cell suspension with the media containing 30% fetal conditioned media and 10% FBS.
8. Cells were plated at a density of 0.5 cells • well−1 • 0.2 ml−1 in 96-well plates.
9. These cultures were examined at frequent intervals using a light microscope and maintained at 37°C and 5% CO_² with a biweekly change of media containing 30% fetal conditioned media and 10% FBS. The cells were maintained in 96-well plates for 2 wk.
10. Wells with cells were scored to determine cloning efficiency.
11. Once cells reached confluence in the microtiter wells, they were trypsinized and transferred to 24-well culture dishes in Primary cell media containing 10% FBS.
12. When the cells had grown again to confluence, they were transferred serially into 12-well, 6-well culture dishes and 25-mm, 75-mm culture flasks, respectively.
13. In all experiments, fibroblasts were studied between the third and the 15th passages.
14. Primary cell medium was changed twice per week, and cells were harvested with trypsin (0.2 g/l) containing EDTA (0.5 g/l).

References
1. Das M, Dempsey EC, Reeves JT, and Stenmark KR. Selective expansion of fibroblast subpopulations from pulmonary artery adventitia in response to hypoxia. Am J Physiol Lung Cell Mol Physiol 282: L976–L986, 2002.
2. Das M, Dempsey EC, Bouchey D, Reyland MR, and Stenmark KR. Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase C isozymes. Am J Respir Cell Mol Biol 22: 15–25, 2000.
3. Das M, Bouchey D, Moore M, Nemenoff RA, and Stenmark KR. Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on G-protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276: 15631–15640, 2001.