Isolation of Choroidal Endothelial Cells and Pericytes

Primary choroidal endothelial cells (CECs) and pericytes were isolated from C57BL/6 mice (4–6 weeks old) as shown in the previous studies^{11 (link),12 (link)}. Briefly, choroidal tissues were digested with 0.05% trypsin (25300120, Gibco, USA) at 37 °C for 30 min. After removing RPE layers, they were digested in 5 ml of digestion solution containing Collagenase II (200 U/ml, 2275MG100, BioFroxx, China) and DNase I (30 U/mL, D7073, Biosharp, China) at 37 °C for 1 h. Then, the cell mixtures were filtered using a 70-μm filter (BS-70-XBS, Biosharp, China). CECs were isolated using anti-CD31 antibody-coated Dynabeads (Invitrogen, USA). The freshly isolated CECs were cultured on collagen IV (17104019, Gibco, USA) coated 6-well plates in the Microvascular Endothelial Growth Medium (EGM2-MV; CC-3202, Lonza) at 37 °C in a humidified atmosphere of 5% CO₂. Primary pericytes were isolated using anti-PDGFRβ antibody-coated Dynabeads (Invitrogen, USA) and cultured in DMEM with 10% FBS at 37 °C and 5% CO₂. CECs and pericytes within the three passages were used for experiments.