Abstract
Purpose: :
Retinal pigment epithelium (RPE) is an important part of blood–retinal barrier that limits access of drugs to the retina and vitreous after ocular injection and from systemic circulation. Therefore, relevant in vitro RPE models are needed for drug delivery studies. The purpose of this study was to test differentiation and barrier properties of polarized ARPE–19 cells grown in filter using different cell culture media.
Methods: :
ARPE–19 culture medium was supplemented with following test ingredients: Taurine, basic fibroblast growth factor (bFGF), all–trans–retinoic–acid (ATRA), bovine retina extract (BRE), rod outer segments (ROS), Na–pyruvate, Na–butyrate, forskolin/IBMX and insulin, dexamethasone, C–vitamin and EGF in combination. The integrity of the cell monolayer was studied by measuring transepithelial resistance (TEER) and by permeability tests (6–carboksyfluorescein and FITC–dextran 40 kDa). The expression of RPE related genes (RPE65, CRALBP, VMD2) were measured by quantitative PCR (TAQMAN).
Results: :
Substantial differences were found in transepithelial resistance levels and in permeability of ARPE–19 model. Permeability of 6–carboxyfluorescein reached almost the levels obtained with isolated choroid/RPE–tissue (2.23+1.06x106 cm/s) (Pitkänen et al 2005) when bFGF (3.37+1.06x106 cm/s) or BRE (3.49 +1.06x106cm/s ) was added to ARPE–19 culture medium. The composition of cell culture medium also affects the expression of RPE specific genes.
Conclusions: :
The integrity of ARPE–19 cell monolayer can be improved by adding bFGF or BRE to culture medium. This effect is reproducible and stable. The expression levels of RPE specific genes can be altered by chancing medium composition.
Keywords: retinal pigment epithelium • retinal culture • gene/expression