Oxygen-Dependent Lipid Profiles of Three-Dimensional Cultured Human Chondrocytes Revealed by MALDI-MSI

[Image: see text] Articular cartilage is exposed to a gradient of oxygen levels ranging from 5% at the surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19–21%), culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes the chondrogenic phenotype. Exposure of cells to various oxygen levels alters their lipid metabolism, but detailed studies examining how hypoxia affects lipid metabolism in chondrocytes are lacking. To better understand the chondrocyte’s behavior in response to oxygen, we cultured 3D pellets of human primary chondrocytes in normoxia (20% oxygen) and hypoxia (2.5% oxygen) and employed matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in order to characterize the lipid profiles and their spatial distribution. In this work we show that chondrocytes cultured in hypoxia and normoxia can be differentiated by their lipid profiles. Among other species, phosphatidylglycerol species were increased in normoxic pellets, whereas phosphatidylinositol species were the most prominent lipids in hypoxic pellets. Moreover, spatial mapping revealed that phospahtidylglyycerol species were less prominent in the center of pellets where the oxygen level is lower. Additional analysis revealed a higher abundance of the mitochondrial-specific lipids, cardiolipins, in normoxic conditions. In conclusion MALDI-MSI described specific lipid profiles that could be used as sensors of oxygen level changes and may especially be relevant for retaining the chondrogenic phenotype, which has important implications for the treatment of bone and cartilage diseases.