A convenient protocol for establishing a human cell culture model of the outer retina.

Abstract

The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch's membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.

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Identifiers

ORCID for Savannah A. Lynn: orcid.org/0000-0003-2513-3144

ORCID for Eloise Keeling: orcid.org/0000-0003-0399-359X

ORCID for David Johnston: orcid.org/0000-0001-6703-6014

ORCID for Angela Cree: orcid.org/0000-0002-1987-8900

ORCID for David Tumbarello: orcid.org/0000-0002-5169-0561

ORCID for Tracey Newman: orcid.org/0000-0002-3727-9258

ORCID for Andrew Lotery: orcid.org/0000-0001-5541-4305

ORCID for J. Arjuna Ratnayaka: orcid.org/0000-0002-1027-6938

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