Stimulation of corneal endothelial cell proliferation in vitro by fibroblast and epidermal growth factors

Abstract

Bovine corneal endothelial cell culture has been developed to study the factors controlling corneal endothelium proliferation. Cells were prepared by scraping the posterior corneal surface gently with a grooved director. The endothelial segments were then suspended in DME with 10% calf serum and 100 ng/ml of fibroblast growth factor (FGF). After 4 days, corneal endothelial cells attached to the tissue culture dish, giving rise to colonies of small, rapidly dividing cells. Fixed cultures stained with silver nitrate to accentuate the inter-cellular function revealed the closely apposed nature of the polygonal cells within the monolayer, and the ovoid nucleus positioned eccentrically in each cell. This morphology is similar to that observed with the corneal endothelium in vivo. The ultrastructure of the bovine corneal endothelial cells grown in vitro was typical of cells with high metabolic rates. Cells had numerous mitochondria, polysomes and microfilament bundles. Ultrastructure and histochemical staining of the culture with alcian green demonstrated that the cells maintained in culture produced extracellular collagenous material. Both fibroblast growth factor (FGF) and epidermal growth factor (EGF) are mitogenic in vitro for endothelial cells derived from bovine corneas. Cells maintained at low density in the presence of 5% bovine plasma divided with a doubling time of 48 hr, but addition of either EGF or FGF reduced the cell doubling time to 20–24 hr. The final cell density reached in the presence of either growth factor was ten times that of cells maintained in 5% plasma alone. In the presence of 10% calf serum, corneal endothelial cells reached a final density identical to that obtained in 1% serum plus FGF and addition of FGF to 10% serum resulted in a final density three times higher than that observed with 10% serum alone. Inclusion of FGF in DME medium containing serum permitted cloning of corneal endothelial cells from cultures seeded at low density (0·6 cells/cm 2). The half-maximal response for the stimulation of proliferation with EGF was seen at 1·5 × 10 −10 m and with FGF at 6 × 10 −10 m. Both EGF and FGF stimulated DNA synthesis in resting corneal endothelial cells, with EGF effective from 1·5 × 10 −15 to 1·5 × 10 −13 m and FGF effective from 7 × 10 −12 to 7 × 10 −9 m. Autoradiography demonstrated that FGF and EGF, like serum, stimulated the cell population as a whole to initiate DNA synthesis.