Raw data from each biological condition were processed using HDI software version 1.5 and 1.7 (Waters Corporation, Wilmslow, UK) to provide ion images from a consolidated list of m/z common to the different datasets as well as the unique m/z. Ion images were normalised to total ion current (TIC). Regions of interest (ROIs) were drawn directly from the DESI images that produced a .csv file containing average TIC normalised intensities which was used for statistical analysis using MetaboAnalyst1 and 6.0 (www.metaboanalyst.ca ) 25 to compare between selected lipids that are presented in the two cell lines. Similarly, MS imaging analysis of Caco2/HT29-MTX co-culture was performed using the same criteria of DESI images with the same precursor ions mentioned above. For pixel classification of DESI imaging datasets, a Waters Corporation (WRC, Budapest, Hungary) prototype AMX MS imaging software was used in combination with HDI software.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["MS Imaging -"],"publication":["Mass Spectrometry Imaging of Lipids in a Gut Epithelial Cell Model."],"submitter_affiliation":["University of Manchester"],"submitter_name":["Qianying Xu"],"organism_part":["Cell"],"technology_type":["mass spectrometry"],"disease":[""],"organism":["Caco-2","Co-Culture","HT29-MTX"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS12599"],"author":["Clare Mills. University of Surrey, University of Manchester. University of Surrey, University of Manchester. clare.mills@manchester.ac.uk.","Lee Gethings. Waters Corporation. lee_gethings@waters.com.","Hadeer Matar. University of Manchester. hmatar@bu.edu.sa.","Qianying Xu. University of Manchester, Manchester Insitute of Biotechnology. qianying.xu-2@postgrad.manchester.ac.uk.","Emmanuelle Claude. Waters Corporation. Emmanuelle_Claude@waters.com."],"data_transformation_protocol":["Raw data from each biological condition were processed using HDI software version 1.5 and 1.7 (Waters Corporation, Wilmslow, UK) to provide ion images from a consolidated list of m/z common to the different datasets as well as the unique m/z. Ion images were normalised to total ion current (TIC). Regions of interest (ROIs) were drawn directly from the DESI images that produced a .csv file containing average TIC normalised intensities which was used for statistical analysis using MetaboAnalyst1 and 6.0 (www.metaboanalyst.ca ) 25 to compare between selected lipids that are presented in the two cell lines. Similarly, MS imaging analysis of Caco2/HT29-MTX co-culture was performed using the same criteria of DESI images with the same precursor ions mentioned above. For pixel classification of DESI imaging datasets, a Waters Corporation (WRC, Budapest, Hungary) prototype AMX MS imaging software was used in combination with HDI software.
"],"study_factor":["Ratio","Lipid"],"submitter_email":["qianying.xu-2@postgrad.manchester.ac.uk"],"sample_collection_protocol":["Caco2 and HT29-MTX cells were cultured separately in bulk in 25 cm2 flasks at 37 °C, 5% CO2 for 2-3 days. DMEM complete media supplemented with 20% (v/v) foetal bovine serum, 2% (w/v) L-glutamine, 1% sodium pyruvate, 1% (w/v) penicillin-streptomycin and 0.01% amphotericin B was used as complete culture medium. The passage number used was P9 for Caco2 and P19 for HT29-MTX after thawing. On reaching 80–90% confluency cells were trypsinised and then seeded onto multiple coverslips to generate replicates at a density of 1x105 cells/mL. These cells were either cultured separately or in a co-culture system, with ratios of 90:10, 75:25, 50:50 and 25:75 (Caco2: HT29-MTX cells). For each condition, four biological replicates were prepared. After seeding, each coverslip was placed in the well of 6-well cell culture plates, and incubated at 37 °C, 5% CO2. Media was changed every 48 hours for 21 days until cells were confluent. Coverslips were prepared for DESI MS imaging analysis by rinsing with 150 mM ammonium acetate (pH 7) for 30 seconds before being allowed to dry in the air stream of a biological safety cabinet for 15 minutes. Coverslips were then thoroughly dried using a vacuum desiccator for another 15 minutes before storage at -80 °C until analysed.
"],"omics_type":["Metabolomics"],"histology_protocol":["Staining was performed according to the kit instructions. Briefly the staining solution was prepared by adding 15 μL ethidium homodimer (EthD-1) and 5 µL calcein to 10 ml of DPBS. Cells were rinsed with pre-warmed DPBS (3 ´ 5 min) to remove any residual medium before adding 300 µl of the stain solution and incubating for 30-40 minutes at ambient temperature. Cells were imaged using a fluorescence microscope EVOS FL (Life Technologies Ltd, Paisley, UK), using either a standard fluorescein bandpass filter emission 494 nm (green) for calcein and filters for propidium iodide emission 535 nm (red) for EthD-1. The green (live cells) and red (dead cells) images were then merged using the fluorescence microscope.
"],"preparation_protocol":["Caco2 and HT29-MTX cells were cultured separately in bulk in 25 cm2 flasks at 37 °C, 5% CO2 for 2-3 days. DMEM complete media supplemented with 20% (v/v) foetal bovine serum, 2% (w/v) L-glutamine, 1% sodium pyruvate, 1% (w/v) penicillin-streptomycin and 0.01% amphotericin B was used as complete culture medium. The passage number used was P9 for Caco2 and P19 for HT29-MTX after thawing. On reaching 80–90% confluency cells were trypsinised and then seeded onto multiple coverslips to generate replicates at a density of 1x105 cells/mL. These cells were either cultured separately or in a co-culture system, with ratios of 90:10, 75:25, 50:50 and 25:75 (Caco2: HT29-MTX cells). For each condition, four biological replicates were prepared. After seeding, each coverslip was placed in the well of 6-well cell culture plates, and incubated at 37 °C, 5% CO2. Media was changed every 48 hours for 21 days until cells were confluent. Coverslips were prepared for DESI MS imaging analysis by rinsing with 150 mM ammonium acetate (pH 7) for 30 seconds before being allowed to dry in the air stream of a biological safety cabinet for 15 minutes. Coverslips were then thoroughly dried using a vacuum desiccator for another 15 minutes before storage at -80 °C until analysed
"],"study_design":["HT29","desorption electrospray ionisation mass spectrometry","Caco-2"],"curator_keywords":["HT29","desorption electrospray ionisation mass spectrometry","Caco-2"],"mass_spectrometry_protocol":["Dried cell cultures grown on coverslips were mounted on microscope glass slides using double sided tape. Slides were scanned using an Epson perfection V600 photo scanner. Scanned images were imported and the area where cells were confluent selected using the co-registered photographic image of the samples in High-Definition Imaging (HDI) version 1.5 and version 1.7. Imaging experiments were carried out on a DESI (Prosolia, USA) mounted on a Xevo-G2-XS quadrupole-time of flight (QTOF) mass spectrometer (Waters Corporation, Wilmslow, UK). The DESI spray was composed of a solvent mixture of 98:2% MeOH: water (v/v) delivered at a flow rate of 2 µL/min with nebulizing gas pressure of 5 bar. The sprayer geometric positions were set that the sprayer was 1.5 mm above sample surface and the distance between sprayer to capillary was 6mm. The source temperature was 150 °C. For both positive and negative ionization modes, the acquisition mass range was 50-1200 m/z. DESI MS imaging experiments were performed using the scan rate of 2 scan/ second in positive mode and 4 scan/ second in negative mode. The X and Y pixel sizes were set at 50 μm.
Selected precursor ions in both positive and negative ionisation modes were further analysed using MS/MS. The experiments were carried out on a DESI-XS (Prosolia, place, USA) mounted on a Xevo-G2-XS Q-TOF mass spectrometer (Waters Corporation, Wilmslow, UK). Spray conditions were the same as DESI imaging. Spectra were visualised using MassLynx ver 4.2 (Waters Corporation, Wilmslow, UK).
"],"metabolite_name":["TG 61:0","TG 63:3"],"additional_accession":[]},"is_claimable":false,"name":"Mass Spectrometry Imaging of Lipids in a Gut Epithelial Cell Model","description":"Scope: The Caco2/HT29-MTX co-culture system is widely used as a cell model of the intestinal epithelium. Although the gut epithelium plays an important role in the uptake of free fatty acids and the resynthesis of triglycerides, the lipid distribution profile of the two-dimensional co-culture system is not well understood. Desorption electrospray ionisation (DESI) mass spectrometry (MS) imaging is a mass spectrometry (MS) technique which has been widely used to study the main classes of lipid molecules on different tissue. This has been used to map the lipid distribution in the Caco2- HT29-MTX co-culture system.
Methods and results: Caco2 and HT29-MTX cells were seeded on coverslips either singularly or as co-cultures at different ratios. Cells were cultured for 21 days before MS imaging using a DESI source in both positive and negative ionisation modes, the identity of selected lipid being confirmed using tandem MS. Although many lipids were common to both cell lines, there were distinctive patterns in the lipidomes. Thus, the lipidome of Caco2 cells was more heterogeneous and richer in cholesterol esters and certain triglycerides whilst HT29-MTX cells has a distinctive lipidome including phosphatidylethanolamines and odd chain phosphatidylcholines and C17 fatty acids.
Conclusion: DESI-MS imaging has shown that Caco2 and HT29-MTX cells have distinctive lipidomes which are still evident when the cells are co-cultured. It has potential to both allow further validation of these widely used cell models and provide insights into how dietary components may modify lipid metabolism in future.
","dates":{"publication":"2025-06-14","submission":"2025-06-14"},"accession":"MTBLS12599","cross_references":{}}