*Purpose: Normothermic machine perfusion (NMP) has gained increasing traction as a method to assess the viability of high-risk donor livers. To avoid post-transplant biliary complications, cholangiocellular viability is currently assessed by point-of-care analysis. The aim of this study was to investigate the underlying, subclinical molecular changes in bile of NMP assessed livers through an in-depth proteomics analysis.

*Methods: Bile samples from 30 NMP perfused livers were collected at 30mins after start NMP (T-30), at viability assessment after 150mins (T-150), and at the end of perfusion (T-end; 17 transplanted livers only). Proteins were prepared using in-gel digestion and analyzed using label-free data-independent acquisition (DIA) quantitative proteomics (LC-MS/MS).

*Results: A total of 77 samples were analyzed, from which 2350 unique proteins were identified. Longitudinal analysis of bile samples revealed 432 and 207 significantly expressed proteins (p<0.05, >2-fold change) between T-30 vs T-150, and T-150 vs T-end, respectively. String pathway analysis at T-150 shows an upregulation of proteins associated with cellular migration, differentiation and immune responses, and downregulation of intracellular and organelle proteins. Pathway analysis at T-end shows an increase of carbohydrate and lipid metabolism, tissue development and cellular signaling processes, with very few downregulated proteins. Analysis of transplanted vs non-transplanted livers at T-150 identified 206 significantly expressed proteins, with transplanted livers showing upregulation of cellular proliferation, migration and immune response proteins (Figure). ROC analysis identified 4 of these proteins to have a combined AUC of 0.986 at T-150.

*Conclusions: This is the first comprehensive proteomics analysis of bile from human NMP perfused livers. Our analysis highlights large and significant protein changes both over time and between transplanted vs non-transplanted livers. Proteins associated with immune responses, cellular proliferation, and migration upregulated in transplanted livers may suggest a higher cholangiocellular functional and regenerative capacity following the injury associated with warm (re)perfusion after SCS.

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