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Tina storm, Ross Campbell, Iain R Wilson, Arantxa Bolinches-Amoros, Angela J. Russel, Stephen G. Davies, Alun R Barnard, Robert E MacLaren; A semi-automated phenotypic RPE in vitro scratch assay for screening of small molecule compounds able to influence RPE wound healing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6394. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration has detrimental effects on retinal pigment epithelium (RPE) cells in the macular region ultimately leading to debilitating effects on vision and quality of life. Current therapeutic approaches to replenish the RPE cells in this region include transplantation of stem cell-derived RPE cells, however so far only with limited success. In vivo studies have shown that the RPE layer has an innate ability to repopulate atrophic areas although to a very limited degree. The aim of this study was to establish a semi-automated in vitro scratch assay for screening of compounds able to influence RPE wound healing.
Compounds were evaluated based on their ability to influence wound healing in a scratched monolayer of differentiated ARPE-19 cells. Cells were seeded in 96-well plates and differentiated to a biological relevant RPE phenotype. The RPE monolayer was subsequently scratched in a 96-well format (WoundMakerTM, Essen BioScience), treated with Hoechst 33342 and an array of compounds. Initial dimensions, intra-well scratch placement and wound healing was evaluated using the EVOS FL auto 2.0 imaging platform combined with semi-automated image analyses based on a purpose designed algorithm.
The average scratch width was 39.3±4% of the well height. The average scratch was located 18±3% of the well height below the centre of the well. A paired t-test showed no significant differences for scratch width and locations across multiple plates. Visual inspection of semi-automated acquired images showed reliable quality and the purpose designed algorithm for automated scratch segmentation showed consistency with manual visual inspection of analysed images. Wells treated with the TAK1 inhibitor (5Z)-7-Oxozeaenol showed a significant decrease in wound healing rate compared to non-treated (p=0.005) or TGF-β1 (p<0.0001) treated wells (one-way ANOVA), validating the biological relevance and robustness of the scratch assay.
We have established a biologically relevant in vitro assay with highly reproducible scratch introduction, semi-automated image acquisition and analyses. This enables us to evaluate a significant number of chemical compounds in a semi-automated manner increasing the potential to identify novel therapeutics that may enhance the innate ability of the RPE cells to repopulate atrophic areas.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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