Isolation and culture of human pulmonary artery smooth muscle cells ...

Isolation and culture of human pulmonary artery smooth muscle cells (PASMCs)

1. The human pulmonary arteries were opened to expose the endothelial surface, which was removed by gentle scraping with a scalpel.
2. The surrounding adventitia was then carefully dissected from the tunica media.
3. Cells were derived either from explants or after tissue digestion.
4. For the explant technique, the pulmonary arterial media was cut into ∼2-mm cubes and plated onto 25-cm² tissue culture flasks.
5. Explants were left to adhere overnight and then were maintained in primary cell culture, 10% FBS, and antibiotic-antimycotic solution (100 U/ml of penicillin, 100 μg/ml of streptomycin, and 250 ng/ml of amphotericin B). 6. The cells were passed after ∼2 wk into a single 75-cm²flask and grown to confluence in primary cell culture system.
7. For tissue digests, the medial layer was cut into small pieces with a scalpel and incubated in type II collagenase (1,000 U/ml) in serum-free medium at 37°C for 4 h.
8. The action of collagenase was stopped by the addition of primary cell culture system.
9. The cells were then passed through a filter (pore size 100 μm) and centrifuged at 200g for 5 min, then resuspended in primary cell culture system before being plated in 25-cm2 flasks.
10. Subsequent passages were carried out at confluence, dividing one flask into four. Cells were used for experiments between passages 3 and 10.
11. The phenotype of isolated cells was investigated with antibodies to smooth muscle-specific antigens: monoclonal anti-α-smooth muscle actin and anti-smooth muscle myosin.
12. For immunostaining, the cells were grown to subconfluence in eight-well slide chambers.
13. The cells were fixed in acetone at −20°C for 10 min, then washed in phosphate-buffered saline (PBS) for 3 × 5 min.
14. The cells were incubated with primary antibody for 1 h at room temperature, then with anti-mouse FITC-conjugated secondary antibody for 1 h, again at room temperature.
15. Between steps, the slides were thoroughly rinsed in PBS for 3 × 5 min at room temperature.
16. The cells were then mounted in a solution of PBS and glycerol (1:1) and visualized by fluorescence microscopy.
References
Morrell NW, Upton PD, Kotecha S, et al. Angiotensin II activates MAPK and stimulates growth of human pulmonary artery smooth muscle via AT1 receptors. Am J Physiol. 1999; 277: L440–L448.