Medium depth influences O2 availability and metabolism in cultured RPE cells
Structured AbstractO_ST_ABSPurposeC_ST_ABSRPE 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 O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. MethodsWe altered the availability of O2 to human primary RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCR), glucose consumption, lactate production, 13C6-glucose flux, hypoxia inducible factor (HIF-1) stability, intracellular lipid droplets after a lipid challenge, trans-epithelial electrical resistance, cell morphology, and pigmentation. ResultsMedium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. ConclusionsDespite 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 (AMD) 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 O2 availability seen with smaller media volumes.