Isolation and Culture of Human PASMCs

Primary human PASMCs were harvested from explanted lungs of patients undergoing transplantation for PH, or from unused lung tissue from donor control subjects. Briefly, human PAs were digested in Hanks’ balanced salt solution (HBSS) containing 2 mg·mL⁻¹ type II collagenase (Worthington Biochem, Lakewood, NJ, USA) and 20 U·mL⁻¹ DNase. Upon the removal of the endothelial layer, the remaining smooth muscles were digested in HBSS containing 2 mg·mL⁻¹ collagenase, 0.5 mg·mL⁻¹ elastase, and 2 mg·mL⁻¹ BSA at 37 °C to make a cell suspension of PASMCs. Isolated PASMCs were resuspended in DMEM containing 10% fetal bovine serum (FBS), 100 IU·mL⁻¹ penicillin, 100 μg·mL⁻¹ streptomycin and incubated at 37 °C in a humidified atmosphere (5% CO₂ in air). The purity of PASMCs was confirmed by the specific monoclonal antibody against α-SMA. Cell viability (usually >98%) was determined by Trypan Blue exclusion. Cells were used at passages 3–6. In vitro hypoxia experiments were performed in primary human PASMC (PromoCell, C12521, Heidelberg, Germany) obtained commercially. To induce hypoxia, cells were placed into a hypoxia chamber with an atmosphere of 5% CO₂/92% N₂, with an oxygen level at 3% for 48 h [55 (link)].