neural retina removal and retinal pigment epithelium (rpe) cell isolation from porcine eyes for in vitro retinal research

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

The retinal pigment epithelium (RPE) is a monolayer located in the outer retina&#160;between Bruch&#39;s membrane and the photoreceptors1. RPE cells form tight junctions with proteins such as zonula occludens-1 (ZO-1) and possess a distinctive phenotype characterized by pigmentation and hexagonal morphology2,3. These cells contribute to the blood-retinal barrier, thereby supporting photoreceptor health and maintaining retinal homeostasis4,5. Additionally, RPE cells play a critical role in vision by absorbing light and recycling essential components for the photoreceptors6. For instance, RPE65, a protein highly expressed in RPE cells, converts all-trans retinyl esters to 11-cis retinol7,8. Given the multitude of functions performed by RPE cells, their dysfunction is implicated in various diseases, including age-related macular degeneration and diabetic retinopathy9,10. To enhance the understanding of retinal pathologies and develop new treatments, in vitro models of the retina are frequently employed.
To generate representative models of healthy or diseased retinas, it is imperative to use a mimetic RPE cell type. The commercially available ARPE-19 cell line lacks native phenotypes, such as pigmentation, while iPSCs can take months to differentiate11,12,13. Although human donor eyes may be ideal, they are often not readily available to many research labs.
Here, we have devised a method to utilize porcine eyes, which share many similarities with human eyes14, for obtaining primary RPE cells. These primary porcine RPE cells have been utilized in multiple retinal models15,16. Not only are these cells cost-effective, but they also require less time to acquire than iPSCs or donor eyes. Additionally, they exhibit native characteristics, such as pigmentation and microvilli. While similar protocols for porcine RPE extraction exist17,18,19, this straightforward and detailed technique further validates enzymatic dissociation and employs materials commonly found in most cell culture laboratories.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

We engineer disease models to mimic aspects of retinal pathologies. With these models, we can better understand the role of RPE cells in diseases based on their response to physical changes, like when strain occurs on the monolayer or a brex membrane thickens with age. One of the biggest challenges is having a representative cell type.Compared to the commonly used immortalized cell line, the primary cells isolated with this technique make our disease models more realistic to what happens in our eyes. This technique provides us with a method of obtaining high quality cells without requiring extensive differentiation times. Furthermore, we use resources that are commonly available to research labs, which makes it more cost effective.Moving forward, we want to look at how natural changes that happen in your eyes during aging, may lead to the early stages of AMD. We want to better understand those changes so we can identify specific treatment targets.

Neural Retina Removal and Retinal Pigment Epithelium (RPE) Cell Isolation from Porcine Eyes for In Vitro Retinal Research

neural-retina-removal-retinal-pigment-epithelium-rpe-cell-isolation

Research

JoVE Journal

Biology

86 Aufrufe.  Utah State University. Sterilisieren und sezieren Sie zunächst die entkernten Augen, die aus einer USDA-inspizierten Metzgerei stammen, um die Augenmuschel vorzubereiten. Legen Sie die Augenmuschel vorsichtig mit der Innenseite nach oben in die Petrischale, in der sich das Zellsieb befindet. Geben Sie gekühltes DPBS in die Augenmuschel und stellen Sie sicher, dass der Flüssigkeitsstand knapp unter dem tiefsten Punkt des Einschnitts liegt.Suchen Sie unter einer Taschenlampe alle Bereiche, in denen sich die...

Serie: Entfernung der neuralen Netzhaut und Isolierung von retinalen Pigmentepithelzellen (RPE) aus Schweineaugen für die In-vitro-Netzhautforschung

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...