Cultured primary human fetal retinal pigment epithelium (hfRPE) as a model for evaluating RPE metabolism

Adijanto, Jeffrey; Philp, Nancy J.

doi:10.1016/j.exer.2014.01.015

Cited by 63 publications

(73 citation statements)

References 47 publications

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“…RPE have high mitochondrial activity and also appear to have high aerobic glycolysis in cell culture models (Adijanto and Philp, 2014; Kurihara et al, 2016). While cell culture models suggest high glycolytic rates in RPE, it is thought that RPE preferentially passes glucose to the photoreceptors (Strauss, 2005).…”

Section: Retinal Cell-specific Metabolismmentioning

confidence: 99%

Retinal energy demands control vascular supply of the retina in development and disease: The role of neuronal lipid and glucose metabolism

Joyal

Gantner

Smith

2018

Progress in Retinal and Eye Research

131

110

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Section: Retinal Cell-specific Metabolismmentioning

confidence: 99%

Retinal energy demands control vascular supply of the retina in development and disease: The role of neuronal lipid and glucose metabolism

Joyal

Gantner

Smith

2018

Progress in Retinal and Eye Research

131

110

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“…These junctional complexes or tight junctions are composed of zonular occludens-1 (ZO-1), occludin, claudins 1, 2, 5, 12 and AL [1]. The retinal pigment epithelium (RPE) performs several complex functions essential for visual function, namely light energy absorption, photoreceptor outer segments phagocytosis, outer blood-retina barrier, secretion of immunosuppressive and growth factors [1,5]. As outer blood-retina barrier, the RPE prevents the transport of molecules larger than 300 kDa via intracellular mediation [6].…”

Section: Introductionmentioning

confidence: 99%

“…As outer blood-retina barrier, the RPE prevents the transport of molecules larger than 300 kDa via intracellular mediation [6]. To be able to perform its complex functions, RPE cells have unique morphological and functional polarity properties, with an expression and polarized distribution of receptors, transporters, channels and enzymes (many of these are markers of differentiated RPE) [5].…”

Section: Introductionmentioning

confidence: 99%

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

Gonçalves

Rodrigues

Padrão

et al. 2016

Colloids and Surfaces B: Biointerfaces

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This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of retinal pigment epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.5-2.0 swelling/mm3), high mechanical strength (2048MPa) and non-pyrogenicity (2.12EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored. Therefore, the ABC substrates coated with UBM showed a great potential to be applied as cell carriers in RPE transplantation.

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“…A significant advance in the field was the development of human fetal RPE cultures, first reported by the Bok laboratory and subsequently by the Miller laboratory (Hu and Bok, 2001; Maminishkis et al, 2006). These cells have since been extensively characterized by many other groups (Ablonczy et al, 2011; Adijanto and Philp, 2014; Sonoda et al, 2009) and have emerged as a powerful system to study RPE function in vitro . However, they require at least four weeks on semi-permeable membrane supports to become fully differentiated (summarized in Table 1).…”

Section: Introductionmentioning

confidence: 99%

A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

Toops

Tan

Lakkaraju

2014

Experimental Eye Research

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The retinal pigment epithelium (RPE) performs numerous functions that are indispensable for photoreceptor health and vision. This monolayer of cells is also a major site of insult in inherited and age-related macular degenerations. In vitro models of primary RPE such as human fetal and adult RPE cultures have been invaluable for dissecting disease pathways at the cellular and molecular level. However, numerous studies show that it takes over four weeks for human RPE cell monolayers to become fully polarized after plating on semipermeable membrane supports. Poor persistence of transgene expression over this time period critically limits the applicability of human RPE cultures for live imaging studies required to follow dynamic processes like intracellular trafficking and organelle transport that occur over timescales of milliseconds. Here, we provide a detailed three-step protocol for live imaging of polarized primary RPE using high-speed spinning disk confocal microscopy. Step 1: establish porcine RPE monolayers that undergo differentiation within one week after plating on semipermeable membrane supports; step 2: transfect or transduce RPE using either of two different protocols that result in prolonged transgene expression; and step 3: perform multicolor high-speed live imaging of organelle transport in polarized RPE monolayers. Porcine RPE cells and photoreceptor outer segments were isolated from freshly harvested eyes and plated on collagen-coated Transwell® filters to generate polarized monolayers. After seven days, RPE monolayers were highly pigmented, had TER values ≥ 200 Ω.cm2 and cleared outer segments within 5 hours after phagocytosis. These cells expressed RPE65, localized ZO-1 to the tight junction, Na+,K+-ATPase to the apical membrane and acetylated tubulin to the primary cilium. There was an inverse relationship between initial plating density and the time to differentiation. We used nucleofection to express fluorescently tagged genes in RPE cells prior to plating on filters or baculovirus fusion constructs to transfect polarized monolayers. Both these methods resulted in transfection efficiencies over 40% and transgene expression lasted up to 8 days after plating. These filters were imaged by high-speed spinning disk microscopy to follow tubulovesicular trafficking of lysosomes and actin dynamics in the RPE. Four-dimensional image analysis performed using commercially available software was used to analyze live imaging data. In conclusion, this 3-step protocol describes a powerful method to investigate organelle trafficking and function in real time in the RPE that can be used for answering fundamental questions of RPE cell biology and pathobiology.

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Cultured primary human fetal retinal pigment epithelium (hfRPE) as a model for evaluating RPE metabolism

Cited by 63 publications

References 47 publications

Retinal energy demands control vascular supply of the retina in development and disease: The role of neuronal lipid and glucose metabolism

Retinal energy demands control vascular supply of the retina in development and disease: The role of neuronal lipid and glucose metabolism

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

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