All usage of human tissues and cells in this study was in
accordance with institutional review board–approved protocols. Irises
from anonymously donated human eyes (Lions Eye Bank, Portland, OR;
donor age range 16–42; no known history of ocular or cardiovascular
disease), were digested in 0.2% type II collagenase (Sigma Chemical
Co., St. Louis, MO) in cell culture medium (MCDB-131;
Clonetics/BioWhittaker, Walkersville, MD) for 20 to 30 minutes at
37°C, after the iris pigment epithelial layer was mechanically
removed with a cotton swab. After digestion, ECs were purified from
iris stromal cells, by using monoclonal anti-human platelet-endothelial
cell adhesion molecule (PECAM)-1 antibody-coated magnetic beads (Dynal,
Inc., Lake Success, NY), and were cultured in medium MCDB-131
supplemented with 10% fetal bovine serum (FBS; Invitrogen Corp.,
Carlsbad, CA), endothelial cell growth factors (EGM-MV2 BulletKit, with
hydrocortisone omitted; Clonetics/BioWhittaker; complete medium),
gentamicin (10 μg/ml), and amphotericin-B (250 ng/ml; Fungizone,
Invitrogen Corp.). Cultures were trypsin passaged at a 1:3 split ratio
and used in subsequent experiments between passages 3 and 6.