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E.G. Heimsath, Jr., A. Tsin, R. Unda; Effects of High Glucose Treatment on ARPE–19 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3814.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) plays a crucial role in retinal physiology. ARPE–19 is a human RPE cell line established by Dunn, et al. (1996). In this study, we sought to determine how ARPE–19 cells respond when grown in high levels of glucose (18mM) versus physiological levels of glucose (5.5mM).
ARPE–19 cells were obtained from the American Type Culture Center (ATCC) at passage 20. To compare the effect of high glucose on cell number and cell morphology, cells were grown in 24–1ml well plates in high (18mM) and low (5.5mM) glucose in DMEM in 10%FBS for eight days. Viable cell numbers were recorded using the Trypan blue methods and hemocytometer. Cell morphology was recorded by measuring the length and width of each cell from a printed image of a visual field in the inverted microscope. To study the influence of high glucose on VEGF secretion, confluence cells were placed in high and low glucose serum–free DMEM for 5 days. On the fifth day, fresh media was added and media was collected every 4 hours for 24 hours for VEGF level using ELISA assays (R&D Systems Minneapolis, MN). To study the effect of retinoic acid on VEGF secretion, cells were treated with all–trans retinoic acid (ATRA; Sigma–Aldrich, final concentration in media: 1µM) and culture media were sampled every 4 hr for a 24 hr period.
Cells grown in low glucose increased from 2.0x104 to 3.3x104 per ml over 8 days whereas cells grown in high glucose increased to 10.4x104 per ml. RPE cells grown in low glucose had a round shape with a length–to–width ratio of 1.6, whereas cells grown in high glucose had an elongated form with a length–to–width ratio of 3.2. VEGF in the culture media of cells grown in high glucose were significantly higher compared to those grown in low glucose (726 and 475 pg/ml respectively). Cells grown in high glucose treated with ATRA associated with a lower level of VEGF (301 vs. 726 pg/ml control without ATRA). This treatment effect was not observed in cells grown in low glucose (439 pg/ml with ATRA vs. 475 pg/ml control).
ARPE–19 cells grown in high glucose (18mM) had higher cell number, different cell morphology, and high VEGF level (in culture media) than those grown in low, physiological level of glucose (5.5mM). Treatment with 1µM ATRA significantly reduced the level of VEGF in culture media of cells grown in 18mM glucose. However, a similar treatment effect was not observed in cells grown in 5.5mM glucose. Our results show that ARPE–19 cells cultured in high glucose exhibited significantly different growth rate, cell morphology and cell functions in comparison to those cultured in physiological glucose level.
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