Arpe 19

The human RPE cell line, ARPE‐19 that was purchased from Procell Life Science&Technology Co.,Ltd (CL‐0026; China), was cultured in DMEM/Ham's F‐12 containing 10% FBS and 100 U/mL penicillin in a humidified incubator with a 5% CO₂ atmosphere at 37°C. After reaching 80% confluence, ARPE‐19 cells were dissociated using 0.25% trypsin for subsequent experiments. Oleic acid (HY‐N1446; MedChemExpress, China) was dissolved in DMSO at 1000 mM and stored at −80°C. For working medium preparation, an Oleic acid solution was added into the prewarmed medium and was incubated under shaking for 6 h at 37°C. ARPE‐19 cells were treated with Oleic acid (100, 250, 500, 1000 µM) for 24 h to induce a lipid‐overload model (Chang et al., 2020 (link)). The condition of 250 µM Oleic acid for 24 h was selected for treating ARPE‐19 cells. ARPE‐19 cells were washed with PBS to remove Oleic acid before adding EVs to the medium. Different concentrations of EVs (1 × 10⁸ EVs/mL, 1 × 10⁹ EVs/mL, 1×10¹⁰ EVs/mL) were used to treat the injured ARPE‐19 cells.

The human RB cell lines Y79 and WERI-RB-1 (Procell Life Science & Technology Co., Ltd., China) were cultured in the Roswell Park Memorial Institute 1640 (RPMI 1640 (HyClone, USA)), and the normal retinal pigment epithelial cell line ARPE-19 (Procell Life Science & Technology Co., Ltd., China) was cultured in Dulbecco's modified eagle medium (DMEM) (HyClone, USA) and high-glucose medium at 37°C with 5% CO₂. All media were supplemented with 10% foetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (Bioswamp, China).

The adult retinal pigment epithelial cell line-19 (ARPE-19) and the human invasive UVM cell line (C918) were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, Hubei, China). ARPE-19 cell was cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12 Ham’s Liquid media (DMEM/F-12; Cytiva/Global Life Sciences Solutions, Marlborough, MA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), along with 100 U/mL penicillin and streptomycin (Gibco, Carlsbad, CA, USA). C918 cell was cultured in Roswell Park Memorial Institute 1640 liquid media (RPMI1640; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), along with 100 U/mL penicillin and streptomycin (Gibco, Carlsbad, CA, USA). Cells were maintained in an incubator (Thermo Fisher Scientific, Waltham, MA) at 37°C, with 95% humidity, and 5% CO2. Cell culture plates, round coverslips, and centrifuge tubes were obtained from (NEST Biotechnology; Wuxi, China). C918 cells were transfected with the generated small interfering RNAs (RiboBio; Guangzhou, China) targeting gene S100A13 and its control siRNAs, according to the manufacturer’s procedure. The siRNA sequences for the gene S100A13 were ACTCGGAGCTCAAGTTCAA.

Human retinal pigment epithelial cell line (ARPE-19) was purchased from the Procell Life Science & Technology in China (CL-0026) and cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% Antibiotic-Antimycotic (Gibco, USA) at 37°C with 5% carbon dioxide. When the cell density reached 80%, it was washed with PBS (Gibco, USA) and treated with 0.05% trypsin (Gibco, USA) for passage at the proportion of 1:3.
The logarithmic growth phase cells with good growth condition were inserted into 6-well plates with 1.2×10⁶ cells per well. The cells were divided into two groups: the HG treated group and the normal group. The HG treated group was cultured in the medium containing 30mmol/L D-glucose, and the normal group was cultured in the SG medium. The 2.5mL medium was added to each well and cultured at 37°C for 48 hours.

Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells and HEK293T cell lines were purchased from Procell (Wuhan, China), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (containing 5 mM glucose) (Thermo Fisher, Waltham, MA, USA) plus 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (Thermo Fisher) in 5% CO₂ at 37 °C.
To induce diabetic retinopathy model, ARPE-19 cells were incubated with higher doses (15, 25, or 35 mM) of glucose for 0, 24, 48, and 72 h. The 35 mM of glucose-treated cells were regarded as the HG group. Cells were observed under a microscope.