Isolation and Culture of Primary Retinal Müller Cells from Sprague-Dawley (SD) Rats

This protocol describes isolation and the culture of primary retinal Muller cells from Sprague-Dawley SD rats, which can aid retinal research in the scientific community. The protocol covers , retinal dissection, CI extraction and identification, and key control considerations. This protocol establishes an efficient, standardized and costly effective method for extracting and recovering RMCs from your legal SD racks. The RMC model can be used to simulate pathological conditions such as diabetic, the normalcy and to assist drug effects.

[Narrator] To begin pour D-Hank's solution into two 10 centimeter glass culture dishes. After euthanizing and disinfecting the neonatal SD rat, position it on a sterile curved dish. Using tooth tweezers, tear the eyelid skin along the palpebral fissure to expose the rat's eyeball. Hold toothless tweezers open and parallel to the palpebral fissure to press down the orbital. Once the optic nerve is reached and the eyeball is exposed, close the tweezers to lift and extract the eyeball. Now place the eyeball in a glass culture dish with D-Hank's solution. Rinse the eyeball and transfer it to another dish with fresh D-Hank's solution. Then using curved ophthalmic micro forceps, gently fix the region between the cornea and optic nerve to expose the cornea. Pierce the cornea scleral junction using micro corneal scissors and cut along the limbus in a circular fashion. Make two symmetrical scleral incisions approximately two millimeters in length before releasing the forceps and reclamping at the junction of the optic nerve and sclera. Then, use a second forceps to gently press near the optic nerve root directing pressure toward the corneal optic nerve interface. When the lens tissue appears, remove it carefully and continue pressing until the retinal tissue emerges. Using forceps, transfer the separated retinal tissue to another sterile culture dish. Open the culture dish lid and use a pipette with a one milliliter tip to pipette the retinal tissue up and down about 15 times to break it into small pieces. Then, incubate the tissue with one milliliter of 0.25% trypsin at 37 degrees Celsius for five minutes. Remove the culture dish from the incubator and place it on the clean bench. Add two milliliters of complete medium and pipette gently to stop the digestion. Now filter the cell suspension through a 300 mesh nylon screen into a 15 milliliter centrifuge two. Wash the culture dish with prepared PBS and collect the remaining suspension. Then spin the tube at 878 G for five minutes at room temperature. After centrifugation, aspirate and discard the supernatant. Resuspend the pellet in two milliliters of complete medium and centrifuge again at 878 G for five minutes to purify the cells. After discarding the supernatant resuspend the cells in two milliliters of complete medium. Take a T25 flask with three milliliters of complete medium and add one milliliter of the cell suspension. Shake the flask in a cross pattern before placing it in the incubator. After 48 hours of incubation, remove the flask from the incubator and place it on the clean bench. Discard the spent medium and wash the cell adhering surface three times with one milliliter of PBS, which contains 1% penicillin plus streptomycin. Then add five milliliters of fresh, complete medium, and continue the incubation until cell confluency exceeds 90%. Wash the cells three times with one milliliter of PBS containing 1% penicillin streptomycin. Then incubate the cells with one milliliter of 0.25% trypsin EDTA solution for one minute and 30 seconds. Now, observe the flask under an inverted microscope. When the cells appear round, detached, and begin to float, add two milliliters of complete culture medium to the flask to terminate digestion. Then use a pipette to aspirate the cell suspension and transfer the entire cell suspension to a 15 milliliter centrifuge tube. Rinse the flask wall with two milliliters of PBS containing 1% penicillin streptomycin, and add it to the same tube. Centrifuge the tube at 878 G for five minutes at room temperature. Discard the supernatant and resuspend the cell pellet in an appropriate volume of complete medium. Finally, passage the cells at a ratio of one to two or one to three, as required. Second passage retinal Muller cells or RMCs displayed star shaped or spindle shaped morphologies with round or oval nuclei and abundant cytoplasm. Hematoxylin and eosin staining revealed spindle and star-shaped cells with abundant pink cytoplasm and centrally located oval nuclei interconnected by fine filamentous structures. Immunofluorescence staining of RMCs revealed strong red fluorescence in cells labeled for Glutamine Synthetase and Aquaporin-4 and bright green fluorescence for CRALBP, Kir4.1 and Vimentin. NeuN, the negative control was not detected in immunofluorescence analysis confirming the specificity of the RMC isolation. Flow Cytometric analysis showed that 98.7% of the cells were positive for Glutamine Synthetase and 97% were positive for CRALBP, indicating a high purity of the RMCs.