Quantitative lipidomics for three-dimensional cell culture using deuterium oxide metabolic labeling

Three-dimensional (3D) cell culture offers a more physiologically relevant model than traditional two-dimensional culture, yet standardized methods for lipid quantification in 3D systems are lacking. This study presents a novel quantitative lipidomic approach combining 3D culture with deuterium oxide (D₂O) metabolic labeling to provide comprehensive insights into metabolic alterations. Using a hydrogel-based 3D system, we cultured preadipocytes and adipocytes, incorporating macrophage co-culture to induce insulin resistance. Relative lipid quantification was achieved using D₂O labeling for global omics relative quantification (DOLGOReQ). This method enabled the quantification of hundreds of lipids across major categories, including glycerolipids, glycerophospholipids, fatty acyls, and sphingolipids, while also revealing cell-type-specific D-labeling efficiencies. DOLGOReQ analysis revealed that macrophage co-culture significantly reduced long-chain free fatty acids and triacylglycerols (TGs). Quantitative correlation analysis between TGs and free fatty acids indicated that the macrophage-mediated TG reduction stemmed from decreased free fatty acid availability, the precursors for lipid synthesis. Furthermore, macrophages increased D-labeling efficiency, suggesting enhanced lipolysis contributing to TG reduction. DOLGOReQ not only facilitates relative quantification of lipid changes but also provides valuable insights into lipid turnover dynamics. These findings establish DOLGOReQ as a powerful tool for investigating global lipid metabolism changes induced by external stimuli in 3D cell culture.