Effects of insulin and EGF on DNA synthesis in bovine endothelial cultures: Flow cytometric analysis

Purpose. To investigate the effects of insulin, epidermal growth factor (EGF), and the corneal storage media-DexSol-at 24 and 48 hours on DNA synthesis in confluent primary cultures of bovine corneal endothelial cells. Methods. Flow cytometry was used to measure changes in DNA synthesis. This technique allows a large number of cells to be counted and sorted into G₁, S, and G₂/M phases of the cell cycle. Results. Changing the normal culture media to DexSol had no effect on the cell cycle at 21 or 48 hours. Tac addition of insulin, EGF, or insulin + EGF to DexSol increased DNA synthesis within 24 hours. The mitotic indices for DexSol, DexSol + insulin, and DexSol + EGF were 0.134 (SE = ± 0.022), 0.207 (±0.027), and 0.205 (± 0.052), respectively. Adding insulin + EGF to the DexSol resulted in the most significant change in S and G₂/M, increasing the mitotic index to 0.300 (± 0.072) (P = 0.0116). At 48 hours, the presence of the growth factors no longer had any effect. Conclusions. Flow cytometry was a useful technique in separating cultured bovine corneal endothelial cells according to their DNA content. Analysis of the cultures after the addition of insulin and EGF showed an increase in DNA synthesis. The synergistic effects of the growth factors on corneal endothelial cells suggest that they stimulate mitotic activity by different mechanisms. The addition of mitogens to eye bank storage media may increase corneal endothelial cell densities in donor corneas.