NOTE: Animal experimentation was approved by national and institutional bioethics committees.

1. ihOEG (Ts12 and Ts14) culture

NOTE: This procedure is done under sterile conditions in a tissue culture biosafety cabinet.

1. Prepare 50 mL ME10 OEG culture medium as provided in Table 1.
2. Prepare 5 mL of DMEM/F12-FBS, as provided in Table 1, in a 15 mL conical tube.
3. Warm both media at 37 °C in a clean water bath, for 15 min.
4. Thaw Ts12 and Ts14 cells vials at 37 °C in a clean water bath.
5. Resuspend and add cells to the DMEM/F12-FBS culture medium prepared in step 2.
6. Centrifuge for 5 min at 200 x g.
7. Aspire the supernatant.
8. Add 500 µL of ME10 medium and resuspend the pellet.
9. Prepare a p60 cell culture dish with 3 mL of ME10 and add the cellular suspension, dropwise.
10. Move to distribute the cells uniformly across the plate.
11. Culture cells at 37 °C in 5% CO₂.
    NOTE: After reaching confluence, at least another passage must be done to optimize cells for coculture. 90% confluence is needed before seeding them on the coverslips for coculture. A confluent p-60 has a mean cell number of 7 x 10⁵ for Ts14 and 2.5 x 10⁶ for Ts12 cell lines. Ts12 and Ts14 cell lines should be passaged every 2–3 days.

2. 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 PLL solution.
2. Wash the coverslips with 1x phosphate buffer saline (PBS), three times.
3. Detach Ts12 and Ts14 ihOEG cells from p60 cell culture dish.
   1. Add 4 mL of DMEM/F12-FBS culture medium (Table 1) to a 15 mL conical tube. Warm at 37 °C in a clean water bath.
   2. Remove the medium from plates and wash cells with 1 mL of 1x PBS-EDTA, 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 medium prepared in step 3.1.
   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.

3. 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 (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 ofreconstituted 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) as provided in Table 1.
2. Sacrifice a rat by asphyxiation with CO₂.
3. Remove the head by decapitation with 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 stay inside (including the pigment epithelial layer).
7. Place the retina in the p60 cell culture dish prepared in step 3.1.1.
8. Transfer the retina to the p60 cell culture dish prepared in step 3.1.2 and cut it with the scalpel in small pieces of an 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 supernatant and to inactivate papain, resuspend the cell pellet in the solution prepared in step 3.1.3. (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 2) 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.

4. Immunostaining

1. After 96 h, fix the cells for 10 min by adding the same volume of 4% paraformaldehyde (PFA) in 1x PBS to the culture medium (600 µL) (PFA final concentration 2%).
2. Remove the media and PFA from the 24-multiwell plate and once again add 500 µL of 4% paraformaldehyde (PFA) in 1x PBS. Incubate for 10 min.
3. Discard the fixer and wash 3 times with 1x PBS for 5 min.
4. Block with 0.1% Triton X-100/1% FBS in PBS (PBS-TS) for 30–40 min.
5. Prepare the primary antibodies in PBS-TS buffer as follows: SMI31 (against MAP1B and NF-H proteins) monoclonal antibody (1:500). 514 (recognizes MAP2A and B proteins) rabbit polyclonal antiserum (1:400).
6. Add primary antibodies to cocultures and incubate overnight at 4 °C.
7. Next day, discard the antibodies and wash the coverslips with 1x PBS, 3 times, for 5 min.
8. Prepare the secondary antibodies in PBS-TS buffer as follows: For SMI-31, anti-mouse Alexa Fluor 488 (1:500). For 514, anti-rabbit Alexa-594 (1:500).
9. Incubate cells with the corresponding fluorescent secondary antibodies for 1 h, at RT, in the dark.
10. Wash the coverslips with 1x PBS, 3 times, for 5 min, in the dark.
11. Finally, mount coverslips with mounting medium (Table of Materials) and keep at 4 °C.
    NOTE: Whenever necessary, fluorescent nuclei staining with DAPI (4,6-diamidino-2-phenylindole) may be performed. Before mounting, incubate the cells for 10 min in the dark with DAPI (10 µg/mL in 1x PBS). Wash the coverslips 3 times with 1x PBS and finally, mount the coverslips with the mounting medium.

5. Axonal regeneration quantification

NOTE: Samples are quantified under the 40x objective of an epifluorescence microscope. A minimum of 30 pictures should be taken on random fields, with at least 200 neurons, to be quantified for each treatment. Each experiment should be repeated a minimum of three times.

1. Quantify the percentage of neurons with axon (SMI31 positive neurite) relative to the total population of RGNs (identified with MAP2A/B 514 positive immunostaining of neuronal body and dendrites).
2. Quantify the axonal regeneration index or mean axonal length (µm/neuron). This parameter is defined as the sum of the lengths (in µm) of all identified axons, divided by the total number of counted neurons, whether they presented an axon or not. Axonal length is determined using the plugin NeuronJ of the image software ImageJ (NIH-USA).
3. Calculate the mean, standard deviation, and statistical significance using the appropriate software.