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P. Geisen, J.R. McColm, B. King, M.E. Hartnett; Characterization of Barrier Properties and Inducible Vegf Expression of Several Types of Retinal Pigment Epithelium in Medium–Term Culture . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2872.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate and compare the characteristics of four different types of retinal pigment epithelial cells (RPE) cultured over one month.
Human cell lines ARPE–19 (ARPE) and D407, primary RPE from C57Bl/6 mouse (mRPE) and primary human fetal RPE (hfRPE) were studied for barrier properties: transepithelial electrical resistance (TER), polarity (Na+, K+–ATPase α–1), permeability (measurement of paracellular movement of sodium fluorescein), ZO–1 and actin staining by immunohistochemistry, and VEGF expression induced in response to exposure to 24 hours of 1% oxygen. VEGF mRNA and protein were measured by real–time polymerase chain reaction (RT–PCR) and ELISA respectively. Madin–Darby canine kidney cells (MDCK) were compared as a non–RPE epithelial cell line.
ARPE at low passage (p15) maintained steady low TER measurements (23 Ohms*cm2) despite forming a monolayer with apical ATPase labeling at 35 days. Mouse RPE maintained a comparable TER for two weeks, but did not localize ATPase. hfRPE had a slow but steady increase in measured TER, but did not have apical localization of ATPase in cultures grown for one month. D407 maintained a low TER (6 Ohms*cm2) and did not localize ATPase. Paracellular permeability reflected barrier integrity measurements in that high TER measurements related directly to low permeability. All human RPE expressed VEGF mRNA and protein. ARPE in 21% oxygen expressed high levels of VEGF protein in media and cell lysates (777.2; 54.4 pg/mg protein, respectively), whereas hfRPE and D407 produced significantly less VEGF compared to ARPE (media: 5.7 [p=0.001], 323.6 pg/mg protein [p=0.01]; lysate: 0 [p<0.001], 3.5 pg/mg protein [p<0.001], respectively). After exposure to 1% oxygen, VEGF protein expression was significantly increased in hfRPE and D407 but not in ARPE.
RPE change from their in vivo state once plated in tissue culture. Primary RPE and those from cell lines have different responses to medium–term culture or after hypoxic stress. Consideration of these differences may be useful in choosing a cell type for individual studies.
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