Deidentified human corneas were obtained from the Center for Organ Recovery and Education (Pittsburgh, PA). Donor human corneas including scleral rim and TM not usable for transplantation were used for experiments. Cells from three donors at 23, 41, and 55 years of age were used in the experiments shown. For each cell population, every experiment was repeated at least once. After careful removal of the iris, a cut was made through the inner edge of Schwalbe's line and the TM tissue was peeled off. We processed TMSCs as either explant culture or dissociated cell culture. For explant culture, the tissue was cut into pieces and put in a 25-cm
2 culture flask. Stem cell growth medium (SCGM) was added and the culture was left undisturbed for 10 to 14 days. For dissociated cell culture, the dissected TM tissue was digested in 0.3 mg/mL collagenase type L (Sigma-Aldrich, St. Louis, MO) in Dulbecco's modified Eagle's medium (DMEM) at 37°C for 20 to 22 hours. After digestion, the cells were filtered through a 70-μm mesh and washed twice with DMEM. Cells were seeded at 2 × 10
4 cells/cm
2 in SCGM. For both cultures, cells were passaged at 80–90% confluency by trypsinization and seeded at 2 to 5 × 10
3 cells/cm
2 in SCGM or seeded for clonal expansion by limiting dilution at 30 cells in a 96-well plate (0.3 cells/well). On average, approximately 1% to 10% of the 96 wells had clones with small cells, which were picked up for subcultivation at 2 to 3 weeks after seeding. Among them, approximately a third to a half could be continuously passaged up to 30 to 50 population doublings. At least six clones from three donors were used and repeated in this study. SCGM was modified from a corneal endothelial cell culture medium
15 containing multipurpose reduced-serum media (OptiMEM-1; Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT), 10 ng/mL EGF (Upstate Biotechnologies, Billerica, MA), 100 μg/mL bovine pituitary extract (Biomedical Technologies, Stoughton, MA), 20 μg/mL ascorbic acid, 200 μg/mL calcium chloride, 0.08% chondroitin sulfate (Sigma-Aldrich), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL gentamicin (Sigma-Aldrich). Primary TM cells were cultured in DMEM without FBS or any growth factors.