Culture à long terme et sans sérum d’explants organotypiques de la rétine de souris avec un épithélium pigmentaire rétinien intact

We described a technique for the isolation of the mouse neural retina together with its retinal pigment epithelium.The retina can be cultured for at least two weeks, with normal development as it would in vivo, retaining the typical tissue architecture and morphology.Organotypic retinal explant cultures make it possible to investigate and manipulate the retina in vitro, under entirely controlled conditions.Avoiding the use of antibiotics and serum.Organotypic retinal explant cultures can be employed to model a variety of retinal diseases, including retinitis pigmentosa and diabetic retinopathy.Explant cultures are also useful for pre-clinical development of novel treatments for retinal diseases.Demonstrating the procedure will be Soumaya Belhadj and Arianna Tolone, two PhD students from my laboratory.Begin by incubating the enucleated eyes in BM for five minutes at room temperature.Then, transfer the eyes into preheated BM with 0.2%Proteinase K, and incubate them at 37 degrees Celsius for fifteen minutes.To inactivate Proteinase K, transfer the eyes to BM containing 20%fetal calf serum, and incubate for five minutes at room temperature on a cool pack.Transfer the eyes to a Petri dish containing fresh BM and aspeptically dissect the eyes under the stereoscope as soon as possible.Using forceps, hold the eye from the optic nerve and make a small incision in the cornea with fine scissors, creating two edges.Peel the cornea, choroid and the sclera gently from these edges using two pairs of fine forceps, or by inserting one of the scissor blades into the edge opening.Grasp the lens with fine forceps and pull to extract it from the eye cup.Using a second pair of forceps placed perpendicularly to the first.Remove the vitreous and the ciliary body attached to the retina.Cut the retina perpendicular to its edges at four points, creating a four leaf clover shape.Hold the retina in a hanging drop of medium using a 1 mL pipet tip with broadly cut base.Transfer it to a culture dish insert placed in a 6-well culture plate, with the RPE layer facing the membrane.Remove the excess medium from the insert, and add 1 mL of CM per well from the sides.Incubate the dish in a sterile incubator at 37 degrees Celsius with 5%carbon dioxide.Avoid submerging the retina in the medium as it will reduce oxygenation and cause tissue degeneration.Keep it at the air-liquid interface.Change the medium every second day by discarding 700 microliters of medium, and replacing it with 900 microliters of fresh medium.After culturing the explants, fix them with 4%paraformaldehyde for 45 minutes.Then perform gradual sucrose cryo protection.When finished, cut the membrane around the retinal explants.Embed both the membrane and the retinal tissue in the medium for frozen tissue, and freeze using liquid nitrogen.Dissected and cultured retinal explants preserve their normal tissue architecture, with distinct layers shown in RD1 mutant and wild-type animals by nuclear staining with DAPI in blue, Rod photo receptors in red, and M