Abstract
In vitro cell models of the gut epithelium, particularly those based on the Caco-2 and HT29-MTX cell lines, play an important role in studying the uptake and metabolism of nutrients and pharmaceuticals. Previous studies using mass spectrometry imaging have shown a distinctive lipidome signature for these cells, alone and in coculture, although only limited information on lipid identities was obtained. A novel method employing limited proteolysis for sampling live, adherent cells using an automated capillary extraction workflow was developed which achieved single-cell sampling of Caco-2 cells although only clusters of HT29-MTX cells could be sampled due to mucus secreted by these cells. The lipidomes of the cell samples were mapped using LC-MS/MS and approximately 150 lipids were putatively identified. Further analysis of these data confirmed the distinctiveness of the Caco-2 and HT29-MTX cell lipidomes. Cell-to-cell heterogeneity was observed, especially in the Caco-2 cells, which may be indicative of variation in their differentiation state. Metabolic pathway analysis showed the distinctive lipidome of Caco-2 cells related to increased glycerol-3-phosphate pathway activity involved in di- and tri- glyceride synthesis. In contrast, HT29-MTX cells exhibited a more active phosphatidylcholine metabolism, related to their mucus-secreting capability. Future studies will explore wider application of the sampling procedure outlined here for single cell lipidomics of other adherent cell lines. Advances in lipidomics analysis at a cellular level highlight important metabolic differences between Caco-2 and HT29-MTX cell lines which are widely used to study gut uptake and metabolism of nutrients and pharmaceuticals.