Isolation of Primary Mouse Retinal Glial Müller Cells

This manuscript describes a detailed protocol for isolating retinal glial Müller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of Müller cells.

Muller cells are the principal glial cells of the retina. They perform a very important role to maintain the highly metabolically active neurons of the retina. Our lab established a technique to isolate Muller cells from mouse eyes. This technique will enable studying the role of Muller cells in different retinal disease, understanding the pathogenesis of diseases, and also development of therapeutic targets.

[Narrator] Ethically euthanize the mouse according to your institution's approved methods. Enucleate the eyes by pressing a 5/45 style tweezer on the orbital area to induce proptosis, followed by extraction of the eye by positioning the arms of the tweezers at the posterior of the eye, followed by gently pulling from the orbital area. Place the eyes in 10 mLs of the Muller cell eye solution and allow them to sit overnight, or for about 18 hours at room temperature. After 18 hours, decant the eye solution by pouring it out of the tube to be discarded. Wash the eyes with warmed phosphate buffered saline or PBS. Decant the PBS again by pouring it out of the tube to be discarded. Then place the eyes in 100 millimeter Petri dish containing 10 mLs of the trypsin solution. Place the dish in an incubator and incubate for 1.5 to 2 hours at 37 degrees Celsius and 5% CO2. After incubating, transfer the eyes to a 60 millimeter Petri dish containing roughly five mLs of complete primary Muller cell growth media. It is worth noting that the video was recorded outside the hood for better visibility and clear demonstration. For actual experiments, they should be conducted in a laminar flow hood as shown. Place the eyes under a dissecting microscope and begin dissecting. Use M5S style tweezers and Vannas scissors to remove connective tissue, extraocular muscles, and the optic nerve. Here, the optic nerve is cut with Vannas scissors. Grasp the eye with M5S style tweezers and make an incision in the ora serrata section with Vannas scissors or the needle of a syringe. Grasp the incision with two M5S style tweezers and begin to pull or tear the cornea from the posterior part of the eye. Pull in small increments to ensure that the integrity of the retina remains intact. Here's a demonstration on how to remove the cornea from the posterior part of the eye cup. Once the lens and neural retina are exposed, remove the pigmented layer of the eye, which likely has the iris adhered to it. Then remove the neural retina from the lens. Remove any pigmented tissue from the neural retina to enhance the purity of the isolation. Given the sticky nature of the neural retina, it will be unlikely you'll remove all the pigmented tissue. Collect all the neural retinas and tear them into smaller pieces. Plate the neural retinas in a T25 flask containing four mLs of complete primary Muller cell growth media. Finally, place the flask in an incubator and incubate at 37 degrees Celsius and 5% CO2. Panel A shows a healthy Muller cell culture at passage zero and passage one. The lack of pigment indicates the absence of RPE cells, which is the primary contaminate in the isolation. This is reinforced in panel B with RP65, an RP specific marker.