Abstract
Purpose :
The retinal pigment epithelium (RPE) has a central role in maintenance of the outer blood-retinal barrier (oBRB). The oBRB efficiently limits drug access to the retina from systemic circulation (i.e. choroidal blood). The purpose of this study was to investigate the permeability, tight junction proteins and oBRB properties of a novel in vitro RPE cell model, derived from human induced pluripotent stem cells (hiPSC).
Methods :
hiPSC-derived RPE cells (PCi-RPE1426, Phenocell, France) were cultured on permeable Transwell® cell culture inserts and the apparent permeability coefficient (Papp) values for standard molecules were determined. Expression of the tight junction proteins, occludin and ZO-1, was characterized in the PCi-RPE1426 model by immunostaining. In addition, tert-butyl hydroperoxide (tBHP)-induced oxidative stress in PCi-RPE1426 cells was assessed by using a resazurin cell viability assay.
Results :
The experimentally determined Papp value for the low permeability standard molecule 6-carboxyfluorescein was 1.0×10-6 cm/s, representative of a tight paracellular barrier between PCi-RPE1426 cells. The dynamic range was >14 in PCi-RPE1426 indicating high discrimination between high and low Papp values. Furthermore, PCi-RPE1426 cells showed low susceptibility to tBHP-induced oxidative stress (IC50 = 2.2 mM).
Conclusions :
We provide evidence that PCi-RPE1426 cells form differentiated cell monolayers with intercellular tight junctions characteristic of the oBRB. This correlated with a decreased Papp for the low standard molecule, representing a 3-7 fold tighter barrier compared to commonly used ARPE-19 cells. In addition, PCi-RPE1426 cells were able to tolerate high levels of tBHP-induced oxidative stress suggesting a potent endogenous antioxidant defense system.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.