To begin, prepare the wash buffer by supplementing Hank's Balanced Salt Solution with 10 millimolar heaps buffer. Store the buffer at four degrees Celsius until use. Prepare retinal pigment epithelium or RPE medium in DMEM and prewarm to 37 degrees Celsius in a water bath.
Lay the euthanized mouse flat on its side with the eye facing upwards. Place the index finger above the eye and the thumb below it. Apply gentle pressure on the bone structure around the eye to cause the eyeball to protrude.
Insert the tip of slightly open scissors under the eye and gently rotate the wrist away from the eye 90 degrees until the eye detaches from the socket. Immediately place and roll the eye in 70%ethanol for five seconds. Then transfer the eye to a wash buffer kept on ice.
Under a dissecting microscope, place the eye in a Petri dish filled with wash buffer and strips of soaked gauze. Stabilize the eye by holding the optic nerve with tweezers and using a surgical knife gently make an incision at the level of the Ora Serrata. Insert Vannas Scissors into the incision and cut around the circumference of the Ora Serrata until the anterior segment and vitreous are removed.
Gently peel away the retina from the eye cup using tethered forceps without disturbing the RPE layer. With the help of scissors, remove the optic nerve and excess connective tissue from the eye cup without puncturing the eye cup. Transfer the eye cup to a 1.5 milliliter microcentrifuge tube containing one milliliter of 0.25%trypsin and 0.02%EDTA.
incubate the eye cup in a 37 degrees Celsius water bath for 10 minutes. Every two minutes, remove the tube and firmly tap the bottom 40 times onto the countertop. After incubation, using AP-1000 pipette gently mix up and down three times to disrupt the eye cup.
To neutralize the trypsin, immediately layer the detached RPE sheets, excluding the eye cup onto 0.5 milliliters of FBS in a 15 milliliter conical tube. Then dropwise, add RPE media onto the layers of FBS and RPE sheets to dilute the trypsin. Centrifuge the RPE at 340G for three minutes.
Discard the supernatant and resuspend the cells in a suitable amount of RPE media for a 24 well plate. Incubate the RPE at 37 degrees Celsius with 5%carbon dioxide for three days. 24 hours after RPE isolation, a nucleate pigmented cells begin to settle without full adherence.
Over the 48 to 72 hours, these cells attach and spread, retaining dark pigmentation in a transparent nucleus. After five days, RPE cells show increased co fluency in start forming cell to cell contacts shaping into a polarized monolayer with hexagonal structures. Isolated RPE showed purity as evidenced by the RPE specific marker and tight junction integrity after six days in culture.
