The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.These studies were supported by the Wellcome-MRC Institute of Metabolic Science (IMS) Metabolic Research Laboratories, Imaging Core (Wellcome Trust Major Award (208363/Z/17/Z)), and the MRC MDU Mouse Biochemistry Laboratory (MC_UU_00014/5). RNAseq was performed by the IMS Genomics and transcriptomics core facility and supported by the UK MRC Metabolic Disease Unit (MRC_MC_UU_00014/5) and a Wellcome Trust Major Award (208363/Z/17/Z). S.V. was supported by BHF (RG/18/7/33636). O.J.C. was supported by a Wellcome Trust PhD studentship. I.K. was supported by a Medical Research Council (MRC) PhD studentship. C.P. was supported by a BBSRC project grant (BB/W005905/1). A.J.M was supported by BBSRC [BB/F016581/1] and BHF [FS/17/61/33473]. D.C.G. is funded by a Sir Henry Dale Fellowship from the Wellcome Trust/Royal Society (210481). J.A.N was supported by a Wellcome Senior Clinical Research Fellowship (215477/Z/19/Z). J.E.H. was supported by a Snow Medical Fellowship. The L.V. lab is funded by the ERC advanced grant New-Chol and the core support grant from the Wellcome Trust and Medical Research Council (MRC) of the Wellcome–Medical Research Council Cambridge Stem Cell Institute. For K.H.F.-W. the work was supported in part by DOD-W81XWH-19-1-0213. C.F. and M.Y. were supported by the MRC Core award (MRC_MC_UU_12022/6). A.V-P. was supported by BHF (RG/18/7/33636) and MRC (MC_UU_12012/2). D.J.F. was supported by a Medical Research Council Career Development Award (MR/S007091/1) and a Wellcome Institution Strategic Support Fund award (204845/Z/16/Z)
