Session: Session 4
Precision Cut Liver Slices analyzed by Mass Spectrometry Imaging: A tool for 3D culture investigation
Precision Cut Liver Slices (PCLS, i.e ultra-thin liver slices that can cultured for several days) are a valuable 3D model for investigating liver pathophysiology and drug efficiency. Mass spectrometry imaging (MSI) is particularly effective for PCLS analysis, as it enables the mapping of hundreds of molecules directly in tissues without labelling and offer insights about microenvironment. Our aim was to develop MSI (Rapiflex, Bruker) workflows for lipidomic and proteomic analysis of frozen PCLS.
After frozen step optimization and embedding assay, to preserve PCLS integrity, slices were thaw mounted on ITO slides for proteomic or lipidomic method optimization. A) For proteomic, 5 washing protocols were evaluated, involving sequential solvents baths before antigen retrieval step. The protocols effectiveness was assessed using optical scans and sensitivity evaluation based on spectra. B) For lipidomic, 4 matrices as 9-aminoacridin (9-AA), 2,5 dihydroxybenzoic acid (DHB), 1,5 diaminonaphthalene (DAN), and α-cyano-hydroxycinnamic acid with various spraying protocols were tested in both positive and negative ionization modes. The evaluation was focused on spectra quality, spatial resolution, and the number of peaks related to biomolecules.
A) The ethanol-based rinse protocol followed by an antigen retrieval step significantly increased sensitivity of peptide detection, particularly for peptides larger than 2500 Da, while preserving the structural integrity of the tissue. B) In negative ionization mode, 9-AA provided the best spatial resolution with the fewest peaks attributed to the matrix. In positive ionization mode, DAN produced the highest peaks number, but many were background overlapping with lipid species peaks. Consequently, DHB was selected for its good spatial resolution and sensitivity.
Optimized MSI workflows offer high sensitivity and spatial resolution, making MSI a robust tool for lipidomic and proteomic analysis of PCLS, suitable for routine integration.