Culturing of Organotypic Retinal Explant Cultures

To study the cytotoxicity of CDs on the retinal tissue, retinas from mice were isolated for culturing for an extended period of time. The detailed protocol is described elsewhere [55], but will be summarized here. Immediately after animal sacrifice, the eyes were enucleated and incubated for 5 min at room temperature (RT) in R16 serum-free culture medium (Gibco, Carlsbad, CA, USA). To promote the removal of the sclera and choroid, the eyes were transferred to a preheated (37 ◦C) solution of 0.12% proteinase K (MP Biomedicals, Illkirch-Grafenstaden, France) and incubated for 15 min. Afterwards, the eyes were soaked in 1:4 mixtures of 10% FBS/medium to stop the protease reaction. The eyes were dissected under sterile conditions. The retina with the retinal pigment epithelium (RPE) attached was isolated and cultured on a Transwell membrane (polycarbonate, 0.4 µm pore size, COSTAR, NY) with the RPE side facing down in a 6-well plate. Then, 1 mL complete medium (CM, R16 medium with supplements; detailed under [55]) was added to each well. The explants were allowed to recover from the explantation procedure in a sterile incubator (37 ◦C, 5% CO2) for 48 h. The CM was exchanged every second day by removing 0.7 mL of the CM in the plate and adding 0.9 mL fresh CM to account for evaporation and conserve neuroprotective agents produced by the retinal culture. At P15, CD and saline solution were applied to the cultures for a 48 h incubation period. Here, 20 µL of isotonic CD solutions (adjusted by NaCl) was carefully placed on the top of the retina to cover the whole tissue. Either 10 or 100 mM CD was used. For fluorescently-tagged CD, 5 mM was applied. Alternatively, a 0.9% NaCl solution was added as a control. All solutions were passed through sterile filters (PES, 0.22 µm, Merck Millipore, Ireland) before being introduced to the culture.
