ARPE-19 and Primary hRPE Cell Culture

This study used the human RPE cell line ARPE-19 (CLR-2302; American Type Culture Collection, Manassas, VA, USA) between 25 and 27 passages and primary human RPE (hRPE) cells (Lonza, Walkersville, MD, USA) between 5 and 6 passages. ARPE-19 cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/100 µg/mL streptomycin (P/S) and passaged at ratios of 1:2 to 1:4 using trypsin–EDTA (Thermo Fisher Scientific, Waltham, MA, USA). Primary hRPE cells were cultured in basal media containing supplements (RtEGM BulletKit; Lonza). For subculture, 2% FBS was added to the media, and the media were replaced with serum-free media after 24 hours. Cells were treated with 10 µg/mL LPS (Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO, USA) with or without fursultiamine–HCl (Toronto Research Chemicals, North York, ON, Canada) at the specified concentrations. Confluent ARPE-19 cells were maintained in a medium consisting of DMEM/F12, 1% FBS, 1.5 mM pyruvate, and 1% P/S for mitochondrial activation, as described previously.¹⁸ (link) The medium was changed every 2 days through days 4 to 6.