Figure 1.

Medium depth limits cellular O₂ uptake by increasing O₂ diffusion distance. (A) Schematic showing the path O₂ must take to get from air, through liquid barriers (culture medium), and to cells at varying apical volumes and constant basal volume. (B) OCR over time for wells at different medium depths (65, 95, 125, 200 µL; n = 6 wells/group, donor 1). Results replicated with two additional hfRPE donors and two iPSC-RPE donors (Supplementary Fig. S2), for a total of five donors across two RPE cell culture types. (C) Average initial steady-state OCR (as designated in B) at each medium depth. (D) [O₂]-dependent or medium volume-dependent changes in HIF-1α, as assessed by Western blot (normalized to GAPDH) (n = 3, donor 2). (E) Medium volume also affects mitochondrial fuel availability. As these fuels are depleted, OCR drops. Plotted is time (in hours) after media change until OCR drops to 50% of steady state (n = 6, donor 1). All cultures are hfRPE. Data are represented as mean ± SEM.