ARPE-19 cells were seeded in a 6-well plate (VWR, Mississauga, ON, Canada. cat # 82050-842) at 600,000 cells/well (60,000 cells/cm2) and reached confluency at ~120,000 cells/cm2. Cultures were maintained for up to 70 days in 2 mL of ARPE-19 culture media that consisted of: DMEM/F-12, HEPES (Thermo Fisher Scientific, Mississauga, ON, Canada. cat # 11330057), 10% FBS (VWR. cat # 97068-085), and 1% Pen/strip (Thermo Fisher cat # 15140122). Media was changed every 48 h by replacing the entire old media volume with 2mL of fresh media. For all experiments, E-RPE cells were cultured in an identical manner to ARPE-19 cells, using ES-RPE culture media that consisted of: 70% DMEM; 30% F12; 2% B-27 supplement and 1% Pen/strip (Thermo Fisher. cat # 11965, 11765, 17504, and 15140122).
To assess the oxygenation status of the cells under the outlined culture conditions, oxygen delivery was calculated using our previously published method [66 (link)]. A RPE oxygen consumption rate of 42 amol∙cell−1∙s−1 was used for the calculation [67 (link)]. We determined that cultured RPE cells should be receiving an adequate amount of oxygen, with a local oxygen concentration of 1.42 × 10−4 mol/L at the cells and a maximum oxygen delivery rate of 191 amol∙cell−1∙s−1.
To assess the oxygenation status of the cells under the outlined culture conditions, oxygen delivery was calculated using our previously published method [66 (link)]. A RPE oxygen consumption rate of 42 amol∙cell−1∙s−1 was used for the calculation [67 (link)]. We determined that cultured RPE cells should be receiving an adequate amount of oxygen, with a local oxygen concentration of 1.42 × 10−4 mol/L at the cells and a maximum oxygen delivery rate of 191 amol∙cell−1∙s−1.
Free full text: Click here
