Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

The overall goal of this procedure is to generate cultures of human fetal retinal pigment, epithelium, or RPE that can be used as a model. In investigations of human eye disease, tissues are received from donors as intact Eye globes. The first step of the procedure is to trim away extra muscle and connective tissue and then open the eye by removing the cornea and lens.

The eye is treated with dispa and flattened, and then the RPE monolayer is peeled away from the choroid.Dissected. RPE are seeded into flasks and grown for several weeks until they reach confluent monolayers and develop pigment. Cell cultures are used in in vitro functional experiments.

Successful in vitro experiments are repeated in preclinical trial animal models where RPE responses can be better evaluated in more complex signaling surroundings. The model demonstrated here will lead to better understanding of RPE physiology and new therapeutic interventions to treat ocular diseases. Hello, my name is Sheldon Miller.

My laboratory is located in the National Eye Institute, which is part of the National Institutes of Health on the Bethesda Maryland campus. Hi, I'm Arki from Miller Lab. Today we will show you a procedure for preparing primary cultures of human fetal retinal pigment epithelial cells.

These cells will be prepared as confluent monolayers that can be mounted in modified sing chambers. We will also use these isolated human RPE cells for studies in biochemistry and genetics. We use this procedure in our laboratory to establish primary cultures of epithelial cells to study the function and regulation of physiological and pathophysiological processes and develop a clinical animal models of diseases.

We focus on the retinal pigment epithelium or RPE, which sits in the back of the eye, separating neural retina and choroidal blood supply. This schematic diagram of the eye shows the location of the RPE sheet in the back of the eye just adjacent to the photoreceptors. The insert shows the cells of the retina with the photoreceptors on the right and the ganglion cells on the left.

This epithelium is metabolically extremely active. It phagocytosis photoreceptors continuously on a diurnal basis, and it stores reis, mizes, and shuttles the visual pigment vitamin A to the photoreceptors. In the visual cycle, it acts homeostatically to maintain the chemical composition and volume of the surrounding extracellular spaces.

A critical property of epithelial is their polarity. The ongoing asymmetric delivery of proteins to the apical and basal lateral membranes is a regulated process that allows the epithelium to carry out the vectorial traffic of metabolites, ions, and fluid from one extracellular space to another. This activity helps epithelium maintain the health and integrity of the neural retina.

The in vitro preparation that we describe here allows the analysis of transport proteins, receptors, and signaling pathways that determine epithelial polarity and function before beginning the dissection. Prepare a 12 well plate in a laminar flow hood with room temperature solutions. Each eye requires one well of 10 x antibiotic antimycotic solution, two wells of one XPBS and one well of two units per milliliter of disc space diluted in 5%RPE medium with serum Fill a silicone padded dissecting dish with one X-H-B-S-S, drain the liquid and transfer each eye into one well of antibiotic antimycotic solution.

Incubate for three to five minutes at room temperature. Avoid applying any excess pressure to the eyeball. Rinse the eye in two successive wells of one XPBS to remove excess antibiotic.

Then use forceps to transfer it to the dissecting dish filled with one X-H-B-S-S under a dissecting stereo microscope. Use iris scissors to remove excess muscle and connective tissue around the eye. Align the eye so that the cornea faces up.

Use two sterile 27 gauge needles to pin the eye by connective tissue without damaging sclera to the silicone pad. In this orientation, use a side port knife to make an incision through the sclera. The cut should be one third of the distance from the eye equator to the anterior surface.

Use iris scissors to continue the cut circularly around the eye to avoid damage to retinal pigment. Epithelium, abbreviated RPE by vitreous Retina attraction. Use the same scissors to cut through the vitreous body to separate front eye flap from eye cup.

Lift the anterior portion of the IOA and discard it. Use forceps to transfer the open eye into the well of dys. Make sure there are no air bubbles in the eye.Cup.

Incubate for 40 to 60 minutes at 37 degrees Celsius with 5%carbon dioxide. Transfer the eye from the dis space back to the dissecting dish, which has been filled with fresh room Temperature one X-H-B-S-S. Position the eye with the cup facing up and secure it with 2 27 gauge needles.

Under the dissecting microscope, use fine forceps to gently lift the partially separated retina with retinal scissors. Cut the retina away from the optic nerve. Discard the retina with the iris scissors.

Make an incision from the periphery of the eye to the optic nerve. Pin the eye out with 5 27 gauge needles. To stretch the RPE layer, use retinal scissors to make a circular cut around the optic nerve.

Separating the RPE layer switching to 250 x magnification or more. Find the edge of the RPE sheet along the cut made by the iris scissors and close to the optic nerve. Use two pairs of forceps to separate the RPE and brus membrane from the choroidal tissue layer.

Try several areas along the cut edge to find where the connection between the RPE and choroid is weakest. Gently peel the membrane back to remove it completely. Place the RPE sheets into a 15 milliliter conical tube filled with room temperature trips in EDTA.

When all the sheets are collected, cap the tube, incubate the tubes in a water bath for 10 minutes At 37 degrees Celsius. Shake the tube vigorously to separate the RPE into small clusters. No dark cell clumps means separation is complete.

Return the tube to the water bath for another five minutes. Use a paster, pipette and gentle suction to remove any undigested tissue leftovers. Spin down the cells in a centrifuge at 280 5G for four minutes.

Remove the supernatant. Bring the final volume up to nine milliliters with 15%RPE medium pipette to resuspend. The cells pipette three milliliters of the cell suspension into a 25 centimeter squared flask.

Add two milliliters of fresh room temperature, 15%RPE medium culture. The cells overnight at 37 degrees Celsius and 5%carbon dioxide. As the cultures mature, their pigmentation will increase after three to four weeks.

Successful cultures will be confluent and uniformly, intensely pigmented. We have just shown you how to prepare human fetal RP primary cultures. So that's it.

Thanks for watching and good luck with your experiments.