Senescent Macrophage Lead to Age Related Macular Degeneration-Like Phenotype in an in vitro 3D RPE/Choroid Model

Abstract

Purpose : Macrophages are integral to maintenance of choroidal function. This study aims to investigate the role of senescent macrophages in inducing advanced wet age-related macular degeneration (AMD) characterized by choriocapillaris neovascularization and functional atrophy of the retinal pigment epithelium (RPE). Utilizing a 3D bioprinted “choroid” with a confluent capillary network, fibroblasts, primary macrophages and polarized RPE monolayer, we aim to model and investigate the mechanisms behind AMD initiation and progression in this 3D Outer Blood Retinal Barrier (3D-OBRB) system.

Methods : Endothelial cells, choroidal fibroblasts, ocular pericytes, and primary macrophages mixed with a collagen-derived gel were 3D-printed on a degradable scaffold to facilitate development of microvascular networks. iPSC-derived RPEs were seeded on the opposite side of the scaffold 7 days after bioprinting. Primary polarized M1, M2, and mixed ratios of M1+M2 macrophages were included in the hydrogel. Confocal microscopy, trans-epithelial electrical resistance measurements (TER), were used to analyze RPE health and structural integrity of choroidal vasculature.

Results : Single (M1 or M2 only) macrophage treatments resulted in increased capillary growth in the choroid space compared to the non macrophage treated control but, didn’t cause any noticeable pathological effect. Mixed macrophage treatments caused increased vascular growth into the 3D-OBRB sub-RPE space, similar in phenotype of type-I CNV, after 5 weeks, with the 2:1 M1:M2 ratio causing the most severe increase. The RPE monolayer also saw decreased TER at the same time point, with 1:2 M1:M2 macrophage treatment causing the biggest decrease. In all cases, macrophages take on “bloated” foam-cell like morphologies, similar to what is seen in patients with advanced AMD. While our bloated macrophages are ki67-/p16+, we are also investigating said macrophages using gene expression analysis to further demonstrate their senesent state.

Conclusions : While healthy macrophages proved to be beneficial during the initial developmental period, development of senescent macrophages coincides with compromised 3D-OBRB physiology. This 3D tissue model allows for the study of ocular immune populations, and their role in ocular health, via a reductionist in-vitro system.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.