The Effect of Glucose on Anoxia-induced Cell Damage in the Cultured Human Retinal Pigment Epithelial (RPE) Cell

Yu, Seung Young; Kim, Dae Ho; Kim, Jae Myung; Kwak, Hyung Woo

Abstract

PURPOSE: It is difficult to make the standard ischemic RPE culture model which is still not established. In ischemic model, oxygen and glucose are very important. To improve experimental model, we evaluate the effect of glucose on anoxia-induced human RPE cell damage.
METHODS
Human RPE cells were incubated with 95% N2/5% CO2 and standard conditions (95% air/5% CO2) at 37degrees C, and various concentation of glucose were added to a glucose-free medium : no glucose, 0.2 mg/ml, 0.4 mg/ml, -2.0 mg/ml. On a daily basis, morphological and proliferation analysis was conducted using the inverted phase contrast microscope. The extent of cell damage was estimated through cell viability analysis. Cell viability was examined using PMS(phenazinemethosulphate)-MTS(3-(4.5 -dimethyl-thiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfo phenyl)-2H-tetrazolium, inner salt)assay after 24, 48, 72, and 96 hours. Single-stranded DNAs were detected with monoclonal antibody to prove cell apoptosis in cell damage.
RESULTS
At slightly low glucose concentration(0.4 mg/ml), the cell viability of human RPE cells cultured at room air was maintained up to 96 hours, whereas that cultured at 95% N2 decreased after 48 hours. At the anoxic condition. the cell viability of human RPE cells cultured with the low glucose concentration (0.2 mg/ml) decreased after 24 hours, but those done with higher glucose concentrations (more than 0.8 mg/ml) were maintained up to 96 hours. In the cell death caused by the anoxic culture, apoptosis was not suggested by single-stranded DNA detection.
CONCLUSIONS
Properly higher glucose concentration on culture medium improve cell viability and reduce the cell damage in human RPE culture in anoxic condition. This result helps to improve experimental ischemic model of RPE cell.