Increased Proliferation and Replicative Lifespan of Isolated Human Corneal Endothelial Cells with l -Ascorbic acid 2-phosphate

PURPOSE: To explore an alternative culture method for human corneal endothelial cells (HCECs) and to examine the effect of l-ascorbic acid 2-phosphate (Asc-2P) on the growth of these cells. METHODS: The influence of various mitogens, extracellular matrices (ECMs), and Asc-2P on growth of cultured HCECs was examined. HCECs were obtained from donors ranging in age from 12 to 74 years, and primary cultures and subcultures were performed with or without Asc-2P. Expanded HCECs were characterized with immunostaining and reverse transcription polymerase chain reaction (RT-PCR) and evaluated for generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) with immunostaining and an enzyme-linked immunosorbent assay (ELISA). RESULTS: Culture with Asc-2P and bFGF on atelocollagen promoted the proliferation of HCECs in both primary cultures and subcultures as efficiently as conventional culture using ECM derived from bovine corneal endothelial cells. Zonula occludens-1, N-cadherin, connexin 43, and Na+/K+-ATPase were localized at plasma membranes of cultured HCECs. mRNAs of the voltage-dependent anion channels (VDAC2 and VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel proteins (CLCN2 and CLCN3) were detected by RT-PCR. During multiple passages, cultures without Asc-2P showed a decrease in growth and irregular cell morphology, whereas cultures with Asc-2P sustained cell growth and maintained the characteristic polygonal morphology. ELISA for 8-OHdG showed that the levels in mitochondrial DNA significantly decreased when HCECs were subcultured with Asc-2P. CONCLUSIONS: Combination of Asc-2P and bFGF on atelocollagen allows successful culture for HCECs. Asc-2P extends the lifespan of cultured HCECs, partly due to protection against oxidative DNA damage.