J Vis Exp. 2018 Apr 12;(134). doi: 10.3791/57084.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane.

Cai H¹, Gong J¹, Del Priore LV¹, Tezel TH², Fields MA³.

Abstract

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for "dry," or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch's membrane pathology is part of this process. Alterations in human Bruch's membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch's membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch's membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch's membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.