Human glial müller and umbilical vein endothelial cell coculture as an in vitro model to investigate retinal oxidative damage. A morphological and molecular assessment

Abstract

The aim of this study was to optimize a coculture in vitro model established between the human Müller glial cells and human umbilical vein endothelial cells, mimicking the inner blood‐retinal barrier, and to explore its resistance to damage induced by oxidative stress. A spontaneously immortalized human Müller cell line MIO‐M1 and human umbilical vein endothelial cells (HUVEC) were plated together at a density ratio 1:1 and maintained up to the 8th passage (p8). The MIO‐M1/HUVECs p1 through p8 were treated with increasing concentrations (range 200–800 μM) of H2O2 to evaluate oxidative stress induced damage and comparing data with single cell cultures. The following features were assayed p1 through p8: doubling time maintenance, cell viability using MTS assay, ultrastructure of cell–cell contacts, immunofluorescence for Vimentin and GFAP, molecular biology (q‐PCR) for GFAP and CD31 mRNA. MIO‐M1/HUVECs cocultures maintained distinct cell cytotype up to p8 as shown by flow cytometry analysis, without evidence of cross activation, displaying cell–cell tight junctions mimicking those found in human retina, only acquiring a slight resistance to oxidative stress induction over the passages. This MIO‐M1/HUVECs coculture represents a simple, reproducible and affordable model for in vitro studies on oxidative stress‐induced retinal damages.