A Comparative Lipidomic Analysis between 2D and 3D Cell Culture of Adipocytes Derived from Mouse Primary Cell

Abstract

Novel Aspect Discovery of lipid biomarkers that distinguish 3D cultured adipocytes from conventional 2D cultured adipocytes via deuterium oxide labeling relative quantification

Introduction Cell culture has been the cornerstone of in vitro experiments to understand the biological response under variable conditions while reducing animal sacrifices. However, the conventional 2D cell culture environment on glass or plastic dish plate is too simple to emulate complex in vivo environment, resulting in different responses between cell and animal experiments. To bridge the gap, in vivo-like 3D cell culture methods has been developed by imitating cell-cell and cell-environment interaction of tissue structure. Numerous studies have shown physiological differences between 2D and 3D cell cultures, but understanding at the molecular level is still lacking. In this study, the Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ) was performed and potential lipid biomarkers were investigated.

Methods The 2D and 3D cultured adipocytes from normal and 5% 2H2O enriched media were counted for normalization to cell numbers. To minimize the difference of lipid amounts in the cell due to their size, the protein from 5% of the cells were extracted and its concentration was measured. A pooled sample was prepared by mixing unlabeled adipocytes to the same amount of protein. Then, the pooled sample was mixed with 2H2O-labeled adipocytes for relative quantification. Lipids were extracted and analyzed with LC-MS/MS followed by measurement of isotopic distributions. And the relative abundances of unlabeled and labeled lipids are calculated from the isotopic envelope of mixed sample. Quantifications of each adipocyte were converted to the quantifications between adipocytes by multiplication.

Preliminary data Sample pooling and normalization based on protein amount were performed to adjust lipid concentration between two cells with different cell sizes, maintaining linearity within dynamic range. The MS / MS fragment ion pattern of each lipid was compared to in-silico library, and mass isotopomers were extracted to obtain relative isotopic abundances. After that, the lipids with poor separation and intensity were filtered out from the subsequent quantitative analysis. We compared the lipid abundance of each adipocyte to the pooled unlabeled sample, and found that adipocyte had higher lipid abundance for overall lipid species. And deuterium incorporation was also higher in adipocytes compared to pre-adipocytes, as our expectation. Interestingly, even though within the same cell line, there were lipidomic differences showing significant quantitative changes between the 2D and 3D adipocytes in the four biological replicates. In the case of pre-adipocyte, quantitative error was increased due to the low lipid abundance and deuterium labeling compared to adipocyte. This led to a decrease in the number of quantifiable lipids. When the lipids of 2D and 3D adipocytes were compared, it was found that triacylglycerol (TG) was the most abundant lipid subclass. And TGs with longer fatty acyl chains were more abundant in 3D adipocytes. In addition, in the co-culture experiment using macrophage, we could explain how the differentiation of adipocyte was affected by macrophage in connection with insulin resistance at the molecular level. Finally, our DOLGOReQ was able to perform global lipid relative quantification with multi-group analysis using pooled sample.