The overall goal of this experimental procedure is to create an ex vivo-extracellular matrix known as the Bruch's membrane culturing system, to that observes the effect of Bruch's membrane on overlaying retinal pigmental epithelial cells. This method could help answer key questions in RPE sub-biology in the AMD etiology field such as whether the changes on aged Bruch's membranes contribute to malfunctions of the overlaying RPE cells. The main advantage of this technique is that it allows researchers to isolate native Bruch's membrane from a human-donor eye of different ages and use them for proteomics study in RPE cell cultures.
Isolating Bruch's membrane and making an actual vivo culture system with minimum damage to the native Bruch's membrane is challenging, but I will show you how in this video. To begin, place a 1.5 square inch gauze soaked in CO2-independent DMEM into a 100-millimeter dish. Then load the eye globe onto the gauze.
Next, use a surgical blade to make an incision through the sclera three millimeters posterior to the limbus. Then, use fine-blade scissors to make a full-thickness incision and circumferentially extend the incision in either direction around the eye. Then, remove and discard the anterior segment including the cornea, lens, and iris.
Also, discard the vitreous and retina. Then, carefully peel the RPE Bruch's membrane choroid complex off the sclera from the periphery to the optic nerve by using a Teflon-coated spatula. This must be done very carefully to not tear the membrane complex.
Now, use fine surgical scissors to make four radial incisions in the sclera and peel away the sclera. Then, cut off Bruch's membrane choroid explant from the optic nerve and transfer this explant to a 60-millimeter dish containing 0.02 Molar ammonium hydroxide in DPBS. To remove the native RPE cells, incubate this explant for 20 minutes at room temperature.
After the incubation, wash the explant three times using DPBS by securing the optical nerve site on the Bruch's membrane with Teflon-coated forceps while flowing solution around the tissue. Next, float the explant, laminin side facing up in carbon dioxide-free media. Make four incisions on Bruch's membrane then transfer an unlaminated hydrophobic, 65 micron-thick PTFE membrane with 0.2 micron pores over the explant.
Set this up with the basal lamina against the PTFE membrane. Remove extra liquid in the dish, then transfer the assembly to a glass microanalysis vacuum filter holder system where it should sit on a fritted glass support. Next, while carefully avoiding contact with basal lamina, flatten the curled edges of the choroidal side using Teflon-coated forceps.
Now, liquefy 15 milliliters of 4%agarose in DPBS and cool it to 37-degrees Celsius. Then, slowly pour the agarose over the choroidal side of the explant while applying gentle suction to keep it flat. Once the gel begins to solidify, transfer it with the membrane to a 60-millimeter dish on ice.
Let the gel solidify for two to three minutes, and then peel off the PTFE membrane. Next, submerge the explant in DPBS and store it at four-degrees Celsius until needed. When culturing the explant on a 60-millimeter dish lined with liquid 4%agarose, make the Bruch's membrane face up.
When culturing using a polystyrene-lined 96-well plate, place the gel-encased Bruch's membrane explant on a flat Teflon sheet with the laminin side face up. Then, using a trephine, cut six to eight six millimeter buttons out of the explant and transfer the tissue buttons to wells loaded with liquid agarose. To begin, set up the eye globe on a DPBS-soaked gauze as before.
Next, make a circumferential incision five millimeters below the iris-sclera contact point, the pars plana, into the subretinal space. Discard the interior segment, vitreous humor, and retina. Next, using a transfer pipette, wash the eye cup with two to three milliliters of cold DPBS.
In total, wash the eye cup three times. Then, to dissociate the RPE cells, treat the eye cup with trypsin for up to 10 minutes. Then, transfer trypsinized RPE cells to a 50-milliliter tube, and to quench the reaction, add 25 milliliters of medium with FBS at 37-degrees Celsius.
Now, centrifuge the tissue and solution at 200g for 10 minutes at 24-degrees Celsius. Then, resuspend the pellet in five milliliters of DMEM complete medium with 15%FBS. Now, count the cells and seat 15, 000 viable RPE cells in 200 microliters of serum-free minimum essential media containing antibiotics onto each button of Bruch's membrane in the 96-well plate.
Culture the cells for at least 24 hours for attachment. Later, gently change the medium to warm complete DMEM and continue culturing the cells. Using this protocol, aged and diseased human Bruch's membranes can be studied by growing RPE cells on them.
Typically, aged explants decrease RPE cell reattachment. RPE cells cultured on aged human Bruch's membrane are also less able to phagocytize rod outer segments, a critical RPE function. Using microarrays, it was found that RPE cell gene expression is different when the cells are cultured on Bruch's membrane from older individuals compared to cells cultured on membrane from younger individuals.
After watching this video, you should have a good understanding of how to isolate Bruch's membrane from human-donor eyes and make explants in which RPE cells can be cultured. Once mastered, this technique can be done in approximately three hours if it's performed properly. While attempting this procedure, it's important to remember not to touch the laminin side of the Bruch's membrane explant with dissecting tools to avoid damaging the surface.
Following this procedure, other methods like RPE phagocytosis and RPE cell gene expression studies can be performed in order to answer additional questions such as whether a Bruch's membrane from a varying aged donor or donors with age-related macular degeneration will affect overlaying RPE cell functions. After its development, this technique paved the way for researchers in the field of age-related macular degeneration to explore the mechanism in etiology of AMD in a Bruch's membrane ex vivo model system.
