Development of a 3D-printed microfluidic chip for retinal organoid-endothelial co-culture

Pathological angiogenesis, such as that observed in wet age-related macular degeneration (AMD), is difficult to reproduce in vitro using human-relevant models. Although organ-on-chip (OoC) systems incorporating retinal pigment epithelium (RPE) and endothelial barriers have been reported, models integrating human retinal organoids with vascular networks remain limited. Here, we present a fully 3D-printed microfluidic platform for co-culture of human induced pluripotent stem cell (hiPSC)-derived retinal organoids containing intrinsic RPE regions with endothelial cells. The device, fabricated from flexible thermoplastic polyurethane (TPU) on a transparent polyvinyl chloride (PVC) substrate, supports three-dimensional co-culture within a fibrin-Matrigel matrix. In this system, endothelial cells formed organized vascular networks that localized around RPE-associated regions of retinal organoids without direct tissue invasion. Organoid-endothelial co-culture resulted in increased VEGF secretion, while exogenous VEGF further enhanced endothelial localization near RPE regions without affecting organoid growth. Functional assays using fluorescent dextran and rhodamine-labeled liposomal nanoparticles demonstrated spatially restricted and time-dependent transport along vascularized regions adjacent to the organoid interface. This retinal organoid-on-chip provides a simple and robust in vitro platform for studying retinal-vascular interactions and vascular-mediated transport processes.