Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of ihOEG (Ts12 and Ts14) for the assay

NOTE: This step must be done 24 h before RGN dissection and coculture.

1. Treat 12 mm Ø coverslips with 10 µg/mL poly-L-lysine (PLL) for 1 h.
   NOTE: The coverslips can be left overnight in the PLL solution.
2. Wash the coverslips with 1x phosphate buffer saline (PBS) three times.
3. Detach Ts12 and Ts14 ihOEG cells from the p60 cell culture dish.
   1. Add 4 mL of DMEM/F12 (basal medium)-FBS (fetal bovine serum) culture medium to a 15 mL conical tube. Warm at 37 °C in a clean water bath.
   2. Remove the medium from the plates and wash the cells with 1 mL of 1x PBS (phosphate-buffered saline)-EDTA (ethylene diamine tetraacetic acid) once.
   3. Add 1 mL of trypsin-EDTA to the OEG cells and incubate for 3–5 min at 37 °C, 5% CO₂.
   4. Collect cells with a p1000 pipette and transfer them to the medium prepared in step 2.1.
   5. Centrifuge for 5 min at 200 x g.
   6. Aspire the supernatant.
   7. Add 1 mL of ME10 medium and resuspend the pellet.
   8. Count the cell number in a hemocytometer.
4. Seed 80,000 Ts14 cells or 100,000 Ts12 cells/well onto the coverslips in 24-well plates in 500 µL of ME10 medium.
5. Culture cells at 37 °C in 5% CO₂ for 24 h.

2. Retinal tissue dissection

NOTE: 2-month-old male Wistar rats are used as RGN source. Two retinas (one rat) for 20 wells of a 24-well cell dish. Autoclave surgical material before use. Papain dissociation kit is commercially purchased (Table of Materials). Follow the provider´s instructions for reconstitution. Reconstitute D, L-2-amino-5-phosphonovaleric acid (APV) in 5 mM stock and prepare the aliquots.

1. On the day of the assay, prepare the following media.
   1. Prepare a p60 cell culture dish with 5 mL of cold EBSS (Earle's balanced salt solution) (vial 1 of the papain dissociation kit).
   2. Prepare a p60 cell culture dish with reconstituted vial 2 (papain) of the papain dissociation kit plus 50 µL of APV. Then, add 250 µL of reconstituted vial 3 (DNase plus 5 µL of APV).
   3. In a sterile tube, mix 2.7 mL of vial 1 with 300 µL of vial 4 (albumin-ovomucoid protease inhibitor). Add 150 µL of vial 3 (DNase) plus 30 µL of APV.
   4. Prepare 20 mL of Neurobasal-B27 medium (NB-B27).
2. Sacrifice a rat by asphyxiation with CO₂.
3. Remove the head by decapitation with a guillotine; place it in a 100 mm Petri dish and spray the head with ethanol 70% before placing it in a laminar flow hood.
4. Cut the rat´s whiskers with scissors so they do not interfere with the eye manipulation.
5. Grip the optic nerve with forceps to pull out the eyeball enough to be able to make an incision across the eye with a scalpel.
6. Remove the lens and vitreous humor and pull out the retina (orange-like tissue) while the remaining layers of the eye (including the pigment epithelial layer) stay inside.
7. Place the retina in the p60 cell culture dish prepared in step 2.1.1.
8. Transfer the retina to the p60 cell culture dish prepared in step 2.1.2 and cut it with the scalpel into small pieces of approximate size < 1 mm.
9. Transfer to a 15 mL plastic tube.
10. Incubate the tissue for 30 min, in a humidified incubator at 37 °C under 5% CO₂, with agitation every 10 min.
11. Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette.
12. Centrifuge the cell suspension at 200 x g for 5 min.
13. Discard the supernatant. To inactivate papain, resuspend the cell pellet in the solution prepared in step 2.1.3 (1.5 mL for 2 eyes).
14. Carefully pipet this cell suspension into 5 mL of reconstituted vial 4.
15. Centrifuge at 200 x g for 5 min.
16. While centrifuging, completely remove the ME-10 medium from the OEG 24 well cell plate (previously prepared in step 1) and replace it with 500 µL of NB-B27 medium per well.
17. Discard the supernatant and resuspend the cells in 2 mL of NB-B27 medium.
18. Plate 100 µL of retinal cell suspension, per well of the m24 plate, onto PLL-treated or OEG monolayers-coverslips.
19. Maintain cultures at 37 °C with 5% CO₂ for 96 h in NB-B27 medium.