Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Place the retina in a previously prepared P-60 cell culture dish with 5 milliliters of cold EBSS. Then, transfer it to P-60 cell culture dish with reconstituted papain plus 50 microliters of APV and 250 microliters of DNase plus 5 microliters of APV.

Cut the retina into small pieces with a scalpel. Transfer the pieces to a 15-milliliter plastic tube, and incubate them for 30 minutes in a humidified incubator at 37 degrees Celsius under 5% carbon dioxide agitating every 10 minutes. Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette. Then, centrifuge the cell suspension at 200 times g for 5 minutes.

Discard supernatant and resuspend the cell pellet in albumin ovomucoid protease inhibitor with 150 microliters of DNase and 30 microliters of APV.

Carefully, add the cell suspension on 5 milliliters of albumin ovomucoid protease inhibitor, and repeat the centrifugation. While centrifuging, completely remove the ME-10 medium from an OAG 24-well plate, and replace it with 500 microliters of NBB-27 medium per well.

Discard the supernatant, and resuspend the cells in 2 milliliters of NBB-27 medium. Plate 100 microliters of retinal cell suspension into each well of the M24 plate onto PLL-treated or OEG monolayer coverslips.

Maintain cultures at 37 degrees Celsius with 5% carbon dioxide for 96 hours in NBB-27 medium.