Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

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 scalpe