M1 and M2 Macrophage contributions towards RPE health and development

Abstract

Purpose : Utilizing a 3D Bioprinted tissue system consisting of vascularized choroidal tissues co-cultured with confluent RPE monolayers, we aim to model the developmental processes of the Outer Blood Retinal Barrier (OBRB). Recently, we included mature macrophages in this system, which become polarized into M1 and M2 macrophages by environmental ques inside tissues likely to fulfil specialized functions . As macrophages are integral to maintaining choroidal function, this study aims to investigate the role of polarized pro-inflammatory (M1) and pro-angiogenic (M2) macrophages, in both the early development and degeneration of the Retinal Pigment Epithelium (RPE) in the 3D-OBRB.

Methods : Endothelial cells, choroidal fibroblasts, and ocular pericytes were encapsulated in a collagen-derived gel and 3D-printed on a degradable scaffold with hydrogels to facilitate microvascular networks. IPSC-RPEs were seeded on the apical side of the scaffold 7 days after biopritning. Finally, primary activated M1 (pro-inflammatory), M2(pro-angiogenic), and M1+M2 polarized macrophages were added to the tissue at the time of printing (at varying concentrations and ratios), as well as at 28 days post printing. Confocal microscopy, quantitative cytokine analysis, trans-epithelial electrical resistance measurements (TEER), were used to analyze RPE health and components.

Results : When M1 and M2 macrophages were added separately on the day of printing or at day 28 post printing, we observed minor changes in TEER. However, these single macrophage populations were able to strongly influence RPE cytokine secretions; with both M1 and M2 separately increasing wet-AMD associated apical secretions. When M1 and M2 macrophages were combined on the day of printing however, TEER of the RPE monolayers degraded after 5 weeks, with increasing TEER decrease associated with increasing M2 content.

Conclusions : The addition of macrophages in this model system influence the growth/survival of our 3D cultured tissues. This 3D-in vitro model may enable investigations into immune mechanisms influencing OBRB development in humans, maximizing ease of access without heavily sacrificing physiological relevance.

This is a 2021 ARVO Annual Meeting abstract.