Preparation of RPE Eyecup Conditioned Media

The RPE eyecups were made as previously reported [19 (link)]. The enucleated eyes were dissected, making RPE eyecups consisting of the RPE monolayer with underlying choroid and sclera with the neuroretina removed. The RPE eyecups were placed into the wells of a 96-well round bottom tissue culture plate (Corning, Corning, NY, USA) containing 200 µL of serum-free media (SFM) (RPMI 1640 supplemented with 10 mM HEPES, 1 mM sodium pyruvate, and nonessential amino acids (Lonza), along with 0.2% ITS+1, 0.1% BSA, and 10 µg/mL gentamycin (Sigma-Aldrich). The cultures were incubated for 24 h at 37 °C in 5% CO₂. The conditioned media (CM) was collected, centrifuged, and the supernatants were used as RPE eyecup-CM.