This article describes a method for isolating retinal ganglion neurons (RGNs) from rat retinas and co-culturing them with olfactory ensheathing glia (OEG) cells. The process involves enzymatic dissociation of the retina, followed by cell culture techniques to assess the neuroregenerative potential of OEGs.
Take a rat's retina, a light-sensitive layer of the eye containing retinal ganglion neurons, or RGNs.
Place the retina in a cold-balanced salt solution to prevent tissue damage.
Transfer the retina to a solution containing protease and DNase and cut it into smaller pieces.
Transfer the contents to a tube and incubate.
The protease breaks down the extracellular matrix and loosens the cells, while DNase degrades any contaminating DNA.
Pipette up and down to disperse cell clumps.
Add an inhibitor solution to stop the protease action.
Centrifuge the cells to settle them. Remove the supernatant and resuspend the cells in a suitable growth medium.
Add this cell suspension to the monolayer of olfactory ensheathing glia or OEG cells, a type of glial cell, and incubate.
The RGNs integrate into the OEG monolayer, establishing the coculture model for assessing the neuroregenerative potential of OEGs.
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.
繁體中文
