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Russell Quinn, Min Jae Song, Anju Singh, Myagmarjav Dashnyam, Devika Bose, Marc Ferrer, Kapil Bharti; Discovering Macrophage Role in Ocular Pathologies Using 3D Bioprinted RPE/Choroid. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4323.
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We previously developed on a 3D-Bioprinted in-vitro outer blood retina barrier that contains an intact, confluent RPE monolayer and a “choroid” with a dense capillary network. In this model, we’re able to study ocular pathologies such as choroidal neovascularization (CNV) and degenerative conditions in the back of the eye. The purpose of this study was to investigate the role of mature macrophages on choroid development and pathology. By adding these components, we aim to increase the accuracy of our in vitro models as well as explore the obfuscating role that these cells play in disease initiation and progression.
A collagen-derived gel is used for encapsulation of endothelial cells, choroidal fibroblasts, and ocular pericytes for bioprinting with additional hydrogels to provide microenvironment conducive for microvascular network formation. We bioprinted a 3D engineered vascularized tissue with a defined geometry on the basal side of a degradable PLGA scaffolds. We then seeded IPSC-RPE on the apical side at 7 days post printing. Finally, primary M1, M2, and M1+M2 polarized macrophages (Dr. Anju Singh/NCATS/NIH) were added to the tissues at set concentrations at days 0, 7, and 21. A combination of confocal microscopy, quantitative western blotting, trans-epithelial resistance measurements, and flow cytometry were used to evaluate the health of these constructs.
We observed M1 and M2 specific phenotypes at various time points, indicating a strong influence on vasculogenesis and angiogenesis in developing vasculature. M2 macrophages promoted tissue growth, and heavily favored angiogenesis at all time points. In contrast, M1 macrophages supported vasculogenesis only when added on day 0. When added to the tissue at later timepoints, M1 polarized macrophages curtailed angiogenesis and lead to vascular degeneration.
We have successfully created an in-vitro construct which allows for investigation of immune mediated development and pathologies of the eye. Future studies will attempt to expand on the mechanisms of macrophage subpopulations in the choroid and RPE.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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