A Quantitative Lipidomic Study for 3D Cell Culture Using Deuterium Oxide Metabolic Labeling

Abstract

Three-dimensional (3D) cell culture is gaining popularity as a more representative in vivo model in contrast to the traditional two-dimensional (2D) cell culture method. Nevertheless, there is no standardized technique for quantifying lipids in 3D cell culture. This study introduces an innovative quantitative lipidomics approach that merges a 3D culture system with deuterium oxide (D2O) metabolic labeling, offering extensive insights into alterations in metabolism. Preadipocytes and adipocytes were cultured within a hydrogel-based 3D cell, alongside co-cultured macrophage cells that triggered insulin resistance. To comparatively quantify lipids, the technique of D2O labeling for global omics relative quantification (DOLGOReQ) was employed. DOLGOReQ facilitated the measurement of numerous lipids spanning key categories like glycerolipids, glycerophospholipids, fatty acyls, and sphingolipids. Importantly, the D-labeling efficiency was notably more consistent in 3D-cultured adipocytes in comparison to their 2D counterparts. By utilizing DOLGOReQ, it was determined that the presence of macrophages led to a significant reduction in free fatty acids and long-chain triacylglycerols (TG). The quantitative relationship between TG and free fatty acids indicated that the decrease in TG was a consequence of lowered free fatty acids, which are precursors for lipid synthesis. Furthermore, the macrophages increased D-labeling efficiency, implying an enhancement in lipolysis and subsequent TG reduction. DOLGOReQ not only allows for the assessment of relative quantitative changes but also offers valuable insights into the dynamics of lipid turnover. These findings underscore the potential of DOLGOReQ as an indispensable tool for scrutinizing global alterations in lipid metabolism prompted by external influences in 3D cell culture investigations.