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Tadashi Senoo, Yoshitaka Obara, Nancy C. Joyce; EDTA: A Promoter of Proliferation in Human Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2000;41(10):2930-2935.
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purpose. To determine whether it is possible to induce proliferation in the
endothelium of older donor corneas by treatment of the intact monolayer
methods. Corneas from donors 52 to 75 years of age were obtained from an eye
bank and were usually cut in quarters to increase sample size. The
effect of EDTA dose (0.02–2.0 mg/ml) and incubation time (6, 30, and
60 minutes) on endothelial cell–cell contacts was evaluated by
staining for ZO-1, a cell junction marker. Cell death was tested by a
commercial live–dead assay. Corneal pieces were incubated for 0, 24,
48, or 60 hours in culture medium (M-199, 10% fetal bovine serum, 10
ng/ml epidermal growth factor, 20 ng/ml fibroblast growth factor)
before EDTA treatment. After treatment, pieces were incubated in the
same medium for 24, 48, 72, or 96 hours to permit cell cycle entry.
Tissue was fixed, stained for Ki67 (a marker for late G1-phase through
the M-phase), and mounted in medium containing propidium iodide to
visualize all nuclei. Confocal images were evaluated by computer (Image
software; NIH, Bethesda, MD) to count Ki67-positive and propidium
results. EDTA released corneal endothelial cell–cell contacts in a dose- and
time-dependent manner. At doses and incubation times tested, EDTA did
not induce significant cell death. Preincubation in culture medium for
24 hours was needed for endothelial cells to efficiently initiate
proliferation in response to EDTA. The endothelium of corneas incubated
in mitogen-containing medium for up to 108 hours without EDTA treatment
did not stain for Ki67. EDTA at 2.0 mg/ml for 60 minutes appeared
optimal and stimulated 16% to 18% of the cells to proliferate.
Ki67-positive mitotic figures were visible 48 hours after exposure to
EDTA. Formation of daughter cells was visible after double-staining for
Ki67 and ZO-1.
conclusions. EDTA released cells from contact inhibition and promoted proliferation
in corneal endothelium from older donors. The authors hypothesize that
corneal endothelium from older individuals divide in situ when exposed
to positive growth factors under conditions in which cells have been
transiently released from contact inhibition.
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