Pharmacologic treatments, enucleation, and separation were performed under dim red light and aseptic conditions. Room temperature was set at 28°C because temperature can modify adhesion between retina and RPE.
3
After enucleation, the cornea, lens, and vitreous were dissected through perilimbal incision, and the optic nerve stump was removed by peripapillar section, allowing detachment of the neural retina without further manipulation. Dissection and separation were made either in Ringer/lactate solution (Fidex, Buenos Aires, Argentina), Hanks balanced saline solution (HBSS) containing 136.9 mM NaCl, 5.37 mM KCl, 1.26 mM CaCl2, 0.49 mM MgCl2 (6 H2O), 0.41 mM MgSO4 (7 H2O), 0.44 mM KH2PO4, 0.34 mM Na2HPO4(7 H2O), 4.17 mM NaHCO3, 5.55 mM glucose, or Ca2+ and Mg2+ free-HBSS (CMF-HBSS) containing 139.06 mM NaCl, 5.37 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4 (7 H2O), 4.17 mM NaHCO3, and 5.55 mM glucose. The RPE-choroid-sclera cup was immersed in RPMI 1640 media supplemented with 25 mM HEPES and l-glutamine (Gibco, Invitrogen, Carlsbad, CA).