Brief protocol for cell isolation:
Cells are isolated from explanted lungs from patients with pulmonary hypertension undergoing lung transplantation at study sites as well as from unused donor lungs. The left lower lobe pulmonary artery (PA) is dissected out and divided into proximal (> 5 mm diameter), intermediate (1~5 mm diameter) and pre-capillary (< 1mm diameter) portions for cell isolation. For main PA cell isolation, approximately 1 cm is dissected at the transplant sites and stored in RAMI1640 buffer at 4C until processed for cell isolation.
For the endothelial cell (EC) isolation, using location-specific procedures, the endothelial surface of each portion of PA is digested with collagenase (Sigma-Aldrich), harvested, and plated on collagen coated plates. Between the primary isolation and the first passage, isolated cells are sorted by CD31-beads (Dynabeads) to capture the ECs. Smooth muscle cells (SMC) are isolated after removing the adventitial layers at each PA location, after which the vessel walls are cut into small pieces, and explanted to the culture plates. The adventitial layer of the each PA location is dissected away and explanted to culture plates.
The cell-specific identity of isolated ECs and SMCs are confirmed by cell specific marker expression and >90% positive populations of the cells are distributed for research.