Twenty-one human corneas were obtained from donors 52 to 75 years
of age (mean age, 65.1 years). Whole corneas were usually cut in
quarters to increase sample size. In some cases, whole corneas were
used as control samples. Corneal pieces were placed endothelial-side-up
in individual wells of a 24-well tissue culture plate (Falcon, Lincoln
Park, NJ). Pieces were incubated for 24 hours in medium-199 containing
10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor (EGF;
Upstate Biotechnologies, Lake Placid, NY), 20 ng/ml fibroblast growth
factor (FGF; Biomedical Technologies, Stoughton, MA), and 50 mg/ml
gentamicin to stabilize the endothelium before study. EDTA (di-sodium
EDTA.2H20) was prepared in Hanks’ balanced salt
solution (HBSS; without calcium chloride, magnesium chloride, or
magnesium sulfate; Life Technologies, Grand Island, NY), adjusted to
pH7.4, and added to the culture medium at a final concentration of
0.02, 0.2, or 2.0 mg/ml. Corneas were treated with EDTA for 10, 30, or
60 minutes and then returned to culture medium for up to 96 hours. EDTA
treatment controls included exposing corneal pieces to all
manipulations and incubation conditions, including 1 hour in HBSS, but
without EDTA. All corneas were maintained at 37°C in a 5% carbon
dioxide, humidified atmosphere until removal for analysis of cell cycle
progression.