Funding information Layton Vision FoundationThe retina is the tissue responsible for light detection, in which retinal neurons convert light energy into electrical signals to be transported towards the visual cortex. Damage of retinal neurons leads to neuronal cell death and retinal pathologies, compromising visual acuity and eventually leading to irreversible blindness. Models of retinal neurodegeneration include 2D systems like cell lines, disassociated cultures and co-cultures, and 3D models like organoids, organotypic retinal cultures and animal models. Of these, ex vivo human retinal cultures are arguably the most suitable models for translational research as they retain complex intercellular interactions of the retina and precisely mimic in-situ responses. In this review, we summarize the distinguishing features of the human retina which are important to preserve in experimental culture, the historical development of human retinal culture systems, the factors affecting ex vivo human retinal culture and the applications and challenges associated with current methods of human retinal explant culture. K E Y W O R D S explant, human, neurodegeneration, post-mortem, retina

Voretigene neparvovec‐rzyl was recently approved for the treatment of Leber congenital amaurosis, and the use of gene therapy for eye disease is attracting even greater interest. The eye has immune privileged status, is easily accessible, requires a reduced dosage of therapy due to its size and is highly compartmentalized, significantly reducing systemic spread. Adeno‐associated virus (AAV), with its low pathogenicity, prolonged expression profile and ability to transduce multiple cell types, has become the leading gene therapy vector. Target diseases have moved beyond currently untreatable inherited dystrophies to common, partially treatable acquired conditions such as exudative age‐related macular degeneration and glaucoma, but use of the technology in these conditions imposes added obligations for caution in vector design. This review discusses the current status of AAV gene therapy trials in genetic and acquired ocular diseases, and explores new scientific developments, which could help ensure effective and safe use of the therapy in the future.

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Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H (CFH) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research.

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment op-tions are limited, partly due to the lack of understanding of AMD pathology and the sparse availability of research models, that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and life-style risk factors contrib-uting to AMD. One of the main genetic risks associated with AMD is located on Complement Factor H (CFH) gene, leading to an amino acid substitution in the FH protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability towards oxidative stress. In this study, we established a novel co-culture model comprised of CFH silenced RPE cells and porcine retinal explants derived from the visual streak of the porcine eyes, closely resembling the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observe that the main changes involve the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE derived FH for retinal homeostasis and provide a valuable model for AMD research.

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Purpose Retinal research relies on animal and in‐vitro models which lack many of the characteristics of human retina. We have instead established a reproducible ex‐vivo model of primary retinal explants derived from human donor eye cups. Methods A dissection strategy performed independently by two investigators was designed to maximize retinal tissue whilst maintaining experimentally reproducible fragments of retina suitable for experimental purposes. The retina was divided into 4 quadrants through the fovea and equivalent distribution of photoreceptors between quadrants was confirmed with CD73 staining in 7 pairs of donor eyes with flow cytometry. In the 8 quadrants from each of the 7 donors, the standard error in proportion of photoreceptors was 0.8–2.3%. Results Cellular composition of free floating retinal explants were followed for 2 months in 10 retinas with quadrant dissection and 8 with random dissection. Explants could be maintained for 2 months with live populations of photoreceptors, ganglion cells and Müller cells detected by flow cytometry. In contrast, amacrine and horizontal cells, decreased by 90% at 7 days. Only quadrant dissection from the same patient showed reproducible and reliable proportions of cell populations between dissections at any time point. There was no statistically significant relationship between the proportion of any cell population and donor age, time after death, time to storage or time in storage. Conclusions The 8 fold quadrant dissection method from a single donor forms an attractive human experimental model for interventional testing in retinal research. We demonstrate the utility of this model in a simple neuroprotection study, showing that insulin protected against CoCl2 induced hypoxia in human photoreceptors.