Primary Culture of Pulmonary Arterial Smooth Muscle Cells

 

1.Primary cultures of Pulmonary Arterial Smooth Muscle Cells (PASMCs) were isolated from human main pulmonary artery.

 

2.A segment of the main pulmonary artery was excised and placed in a sterile 10-cm dish containing primary cell culture media.

 

3.The segment was stripped of adventitia with a sterile forceps.

 

4.The main pulmonary artery segment was then cut longitudinally to open the vessel, and the endothelial layer was removed by gentle rubbing with a cell scraper.

 

5.The vessel was then cut into 2-mm segments, inverted, and placed on a collagen-coated 35-mm tissue culture dish.

 

6.A drop of primary cell culture media containing 10% fetal bovine serum, primary cell culture supplements, antibiotics, and antimycotics was then added, and the cells were grown overnight at 37°C in a humidified atmosphere with 5% CO^₂-95% air.

 

7.The next day, an additional 2 ml of complete medium were added.

 

8.The growth medium was subsequently changed every 2 days.

 

9.When SMC islands could be observed under the microscope, the tissue segment was removed and the individual cell islands were subcloned.

 

10.Identity was confirmed as PASMCs by immunostaining (>99% positive) with SMC actin. This was taken as evidence that cultures were not contaminated with fibroblasts or endothelial cells.

 

11.All cultures for subsequent experiments were maintained in primary cell culture media containing 10% fetal bovine serum, primary cell culture supplements, antibiotics, and antimycotics at 37°C in a humidified atmosphere with 5% CO^₂-95% air.

References

 

1.Chamley-Campbell, J., Campbell, G., and Ross, R.: The Smooth Muscle in Cell Culture, Physiol. Res. 59, 1, 1979.

2.Richardson, M., Taylor, D., Casey, M., MacDonald, P., and Stull, J.: Biochemical Markers of Contraction in Human Myometrial Smooth Muscle Cells in Culture, In Vitro Cell. Dev. Biol. 23, 21, 1987.

3.Eskin, S., Sybers, H., Lester, J., Navarro, L., Gotto, A., and DeBakey, M.: Human Smooth Muscle Cells Cultured From Atherosclerotic Plaques and Uninvolved Vessel Wall, In Vitro 17 (8), 713, 1981