Characterization of in vitro outer blood retinal barrier model

Abstract

Background and aims: The human eye has anatomically closed structure, due to the blood retinal barrier (BRB) which is protecting the photoreceptor cells. It comprises of three layers: retinal pigment epithelial cells (RPE-cells), endothelial cells and a collagenous membrane in between the two. The blood retinal barrier is highly selective in passing molecules between the blood stream and the retina. This closed structure makes the in vivo –study and treatment of the retinal conditions challenging. Even up today, the pathological mechanisms of many ocular pathologic conditions, like the age related macular disease, are not fully understood. Most BRB studies are made with ex vivo tissue samples isolated from animals or human eyes after surgical operations or from cadaveric subjects. In addition, monocultures of cells have been done. However, problems arise with the availability and the resemblance of these models compared to the authentic tissue. So far very little studies have been made towards a trilayered in vitro –model, where both of the cell types are co-cultured. The purpose of this study is to address the need for this type of in vitro –model, by co-culturing the different cell lines and by evaluating the acquired samples.

Materials and methods: In this study three RPE cell lines derived from human embryonic stem cells and one immortalized RPE cell line (ARPE-19) was used. As endothelial cells, commercially available ACBRI181 primary cells were used. The cells were co-cultured with RPE cells on the other side and endothelial cells on the other side of a PET membrane. Additionally, monolayer cultures of single cell type were made for better overall evaluation. The visibly matured samples were characterized by evaluating their barrier properties with electrical resistance meter, their permeability for small sized molecule with Ussing chamber test and spectrophotometry, and finally their polarity and maturity with indirect immunofluorescence and confocal microscope.

Results and conclusions: Based on the results the human embryonic stem cell derived RPE cell lines seemed to perform well in producing a matured and polarized RPE layer. The endothelial cell layer was not uniform in the trilayer co-culture, however it remains under speculation whether that is more typical in the in vivo –situation as well. Nevertheless, the presence of the endothelial cells seemed to improve the barrier properties of the samples, compared to the RPE-monoculture samples. This result was good and expected, yet there was no statistical significance with the result material gathered. The culture samples made with ARPE-19 cell line did not succeed as hoped; they did not resemble RPE-layer. All in all, the results are interesting and encourage continuing this research line more.