Medium Depth Influences O(2) Availability and Metabolism in Human RPE Cultures

PURPOSE: Retinal pigment epithelium (RPE) oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O(2) availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. METHODS: We altered the availability of O(2) to human primary and induced pluripotent stem cell–derived RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCRs), glucose consumption, lactate production, (13)C(6)-glucose and (13)C(5)-glutamine flux, hypoxia inducible factor 1α (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, transepithelial electrical resistance, cell morphology, and pigmentation. RESULTS: Medium volumes commonly employed during RPE culture limit diffusion of O(2) to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O(2) availability decrease acetyl-CoA utilization, increase glycolysis and reductive carboxylation, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. CONCLUSIONS: Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology “under the hood.” As RPE-centric diseases like age-related macular degeneration involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O(2) availability seen with smaller media volumes.