Abstract
Purpose :
Choroidal neovascularization (CNV) is a major cause of blindness during later stages of age-related macular degeneration (AMD). The mechanisms of CNV in AMD remain unclear and realistic in vitro models of retinal tissue are lacking. Usually, Bruch’s membrane (BrM) models overlook key properties of in vivo BrM, such as thickness and nonporous permeability. We propose a more mimetic three-layered microfluidic model of subretinal tissue, consisting of the primary porcine retinal pigment epithelium (RPE), hagfish protein BrM, and human umbilical vein endothelial cell (HUVEC) choroid.
Methods :
Microfabrication techniques were used to create polydimethylsiloxane (PDMS) cell channels. Hagfish protein BrM models were produced and thickness, permeability, and biocompatibility was characterized. Transendothelial electrical resistance (TEER) measurements were taken to verify tight junction formation of RPE on BrM model. The microfluidic device was then constructed by layering a PDMS base layer, cell layer, BrM model, cell layer, and top layer onto a glass microscope slide, using oxygen plasma treatment to bond each layer. HUVECs and RPEs were loaded into the lower and upper cell channels respectively. Media was perfused through the lower channel at 2.88mL/hr and replaced in the upper channel every 24 hours. Immunocytochemical (ICC) staining was used to visualize vascular endothelial growth factor (VEGF), zonula occludens-1 (ZO-1), and angiopoietin-2 (Ang-2). An enzyme linked immunosorbent assay (ELISA) was used to quantify VEGF, ZO-1, Ang-2, and tissue inhibitor of metalloproteinase-1 (TIMP-1).
Results :
PDMS cell channels have 5mm of observation area with a width of 823 ± 8.7mm and thickness of 112 ± 0.4mm. Hagfish protein BrM support RPE tight junction formation and demonstrate a more representative model of in vivo BrM thickness and permeability. The constructed device is expected to show RPE VEGF, Ang-2, and TIMP-1 diffusion through the BrM model to initiate interconnected vascular network formation in the endothelial cells.
Conclusions :
We hypothesize that this in vitro microfluidic model of the subretinal tissue will lead to a better understanding of the mechanisms of CNV initiation and progression in AMD. Diseased conditions can be induced by chemical hypoxia or high-dose VEGF perfusion in RPE channel to monitor changes in protein expression and system behavior.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.