Methods for Growing Retinal Pigment Epithelial Cells: Current Protocols and Future Recommendations (2021)

The retinal pigment epithelium (RPE) is a layer of tissue found in the vertebrate eye between Bruch’s membrane and the photoreceptor layer of the neural retina [1]. It is derived from the outer layer of the optic cup [2], possesses an innate immune system [3], and consists of a monolayer of highly pigmented cells that fit together in a tight matrix (fig. 1) [4]–[6]. The monolayer is often compared to a mosaic or cobblestones in its configuration, while the shape of the individual cells is usually described as polygonal/hexagonal, columnar (aligned perpendicular to the underlying membrane), or “epithelioid”. The cells are strongly polarized[7], with microvilli on the apical surface [8]. Despite its simplicity as a tissue layer, the RPE plays many complex roles in the vertebrate eye, including regulation of retina development [9], [10], absorbing excess light entering the eye to reduce photo-oxidative stress[11], secreting growth factors such as vascular endothelial growth factor (VEGF)[5], [10], [12], mediating the immune response of the eye [13]–[15], transporting metabolites and fluids [16]–[18], and phagocytosing spent rod and cone outer segments [19]–[26]. The RPE also acts as an intermediate for supplying glucose and other vital nutrients to the retina [27] while maintaining a good environment for the photoreceptors [28] and preventing large molecules from entering the eye from the bloodstream [29]. This last purpose designates the RPE as part of the blood-retinal barrier, which is primarily in place to stop particles from entering the vitreous humor and obscuring vision [30]–[32]. While the RPE plays many roles in the eye, its greatest medical significance comes from its involvement in many ocular disorders [33] that can lead to vision loss or blindness, such as retinitis pigmentosa, diabetic retinopathy, West Nile virus, and macular degeneration [12], [34]–[46]. Macular degeneration in particular is currently of great interest to medical and biological researchers. Age-related macular degeneration (AMD) is a disorder of the retina characterized by loss of sight in the center of the visual field, and is the leading cause of vision loss after age 50 in developed countries [47]. AMD occurs in two forms: the exudative or “wet” form, and the nonexudative or “dry” form. Wet AMD (the most severe form, also called choroidal neovascularization) is caused by excessive growth of capillaries from the choroid and into Bruch’s membrane and the RPE in response to production of vascular endothelial growth factor (VEGF)[48]. These fragile new blood vessels exude blood, lipid, and protein below the macula (the central region of the retina, necessary for visual acuity), causing scarring and sudden acute vision loss [49]. Dry AMD (the most common type, also called atrophic AMD or geographic atrophy)[50] is a much slower process, characterized by degeneration of the RPE and later the neural retina [49], [51], [52], which can lead in some cases to tearing of the RPE [53]. Its causes are not fully understood [29] [30], although evidence suggests contributing factors include the accumulation of lipofuscin and other substances in the eye [32], [56]–[61], disruption of autophagy and other processes in the RPE [23], [26], [62]–[64], photo-oxidative stress caused by excess light entering the eye [32], [60], [65]–[71], and other retinal disorders like focal choroidal excavation (FCE)[72].