Abstract
Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.
Highlights
The retinal pigment epithelium (RPE) is a monolayer of highly polarized cells that performs multiple functions to support retinal health, such as phagocytosis of photoreceptor outer segments, autophagy, transporting nutrients and materials for the visual cycle, maintaining the blood–retinal barrier, etc. [1]
RPE degeneration has been identified as a key feature in many retinal diseases, for instance, retinitis pigmentosa, Stargardt disease, and age-related macular degeneration (AMD) [2]
RPE Flatmounts Are Viable after 7 Days in Culture
Summary
The retinal pigment epithelium (RPE) is a monolayer of highly polarized cells that performs multiple functions to support retinal health, such as phagocytosis of photoreceptor outer segments, autophagy, transporting nutrients and materials for the visual cycle, maintaining the blood–retinal barrier, etc. [1]. The limitations of this technique are low transepithelial resistance, lack of pigmentation, and abnormal karyotype [4] Another option is obtaining RPE cells from mouse- or human-induced pluripotent stem cells (iPS-RPE) or embryonic stem cells (ES-RPE) by culturing them in differentiating medium either in dishes or on semi-permeable transwell filters to allow fluid flow and to enhance polarization. Obtaining well-differentiated RPE cells from either iPS cells or ES cells is expensive and time-consuming (typically 6–12 weeks) [6,7], which may diminish enthusiasm for working with them To overcome these difficulties, primary RPE cells isolated and cultured from rodents, pigs, or human eyes are commonly used in many RPE studies. We describe the use of adenovirus vectors to efficiently express genes of interest in RPE flatmounts in vitro, which improves gain- and loss-of-function studies as well as investigations on signaling pathways compared to any other previously described methods
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