A primary retinal pigment epithelial (RPE) cell culture model produces lipid- and hydroxyapatite-rich extracellular deposits characteristic of early stage age-related macular degeneration

Abstract

Purpose : Accumulation of lipid- and protein-rich drusen and basal linear deposits between the inner collagenous layer of the Bruch’s membrane and the basal lamina of the RPE is a hallmark of early age-related macular degeneration (AMD). Hollow spherules composed of lipids and inorganic hydroxyapatite (HAP) have been described within these deposits (PMID 25605911). We report a primary RPE cell culture model capable of producing sub-RPE deposits with these major molecular components.

Methods : RPE cells were isolated from freshly enucleated porcine eyes and cultured on laminin-coated porous Transwell membranes (Millipore). Cells were cultured in Miller medium (PMID 16877436) without addition of photoreceptor outer segments for up to 6 months. Histochemical studies were performed and cultures were examined by light and fluorescence microscopy as well as by scanning (SEM) and transmission (TEM) electron microscopy. Mineral deposition was detected by micro-CT and synchrotron x-ray diffraction (µXRD), trace metal composition was determined using synchrotron x-ray fluorescence (µXRF), and protein and lipid components were identified using secondary ion mass spectrometry (SIMS).

Results : TEM analysis of differentiated and polarised RPE cell monolayers revealed an age-dependent accumulation of diffuse and focal deposits between the RPE cell monolayer and the underlying Transwell membrane. Deposits were highly mineralised and produced diffraction patterns characteristic of the inorganic calcium phosphate hydroxyapatite. Zinc and iron co-localized with hydroxyapatite, but copper signal could not be verified. SIMS analysis confirmed the presence of proteins and lipids in focal deposits.

Conclusions : Healthy primary RPE cell cultures produced focal and diffuse deposits containing lipids, proteins, trace elements and hydroxyapatite, all components of human drusen and basal linear deposits. These deposits formed without the addition of photoreceptor outer segments suggesting that RPE cells are capable of generating nucleation sites for sub-RPE deposit formation, this is consistent with previous in vitro (PMID 21969589) and pathology (PMID 21890786) studies. This cell culture model of early stage AMD provides a novel system for which new therapeutic interventions against early stages of AMD could be trialed.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.