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. 2023 Apr;86(4):439-451.
doi: 10.1002/jemt.24284. Epub 2022 Dec 29.

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

Gloria Astolfi  1 , Carmen Ciavarella  2 , Sabrina Valente  2 , Chiara Coslovi  1   3 , Danilo Iannetta  1   3 , Luigi Fontana  1   3 , Gianandrea Pasquinelli  2   3 , Piera Versura  1   3
Affiliations

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

Gloria Astolfi et al. Microsc Res Tech. 2023 Apr.

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 H2 O2 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.

Keywords: GFAP; HUVECs; MIO-M1 cells; coculture; oxidative stress.

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