Rats with healthy, untreated eyes were killed by exposure to a rising concentration of carbon dioxide followed by cervical dislocation. Immediately, both eyes were enucleated (
Supplementary Fig. S1a) and immersed in ice-cold HBSS containing penicillin (100 U/mL) and streptomycin (100 μg/mL). With an operating microscope, a circumferential incision was made around the limbus (Supplementary Fig. S1b), followed by removal of the anterior segment, lens, and vitreous body (Supplementary Figs. S1c, S1d). With an extremely fine paintbrush, the retina was carefully peeled away from the retinal pigment epithelium (Supplementary Fig. S1e), and a single cut was made at the optic nerve head, thereby severing the retina from the optic nerve (Supplementary Fig. S1f). The retina was dissected radially into four equal-sized pieces (Supplementary Fig. S1g), and the explants were separately transferred onto 12-mm diameter filters (0.4 μm pore, Millipore; Millicell Inc., Cork, Ireland; Supplementary Fig. S1h) with the RGC side facing up. The filters were placed into the wells of a 24-well plate, each of which contained 300 μL of retinal explant media. Based on preliminary experiments (data not shown), two different types of culture media were selected for further analysis. Medium containing normal horse serum (NHS) was produced based on previous reports
19 20 21 22 and consisted of a neuronal growth medium (Neurobasal-A) supplemented with 25% heat-inactivated NHS (Invitrogen Ltd., Paisley, UK),
l-Glutamine (0.5 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL). A serum-free medium (B27/N2) was modified from previous studies
13 14 15 and contained the neuronal growth medium (Neurobasal A) supplemented with 2% B27 (Invitrogen Ltd.), 1% N2 (Invitrogen Ltd.),
l-glutamine (0.8 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL). Retinal explant cultures were maintained in humidified incubators at 34°C and 5% CO
2. Half of the media were changed on day ex vivo (DEV) 1 and every second day thereafter.