<|endoftext|>iporeSigma, Frankfurter, Germany) for cell count and characterization.
Using a gas analyzer, acid-base balance (pH, pCO~2~, pO~2~), electrolytes (K^+^, Na^+^, Cl^−^, Ca^2+^), and metabolites (glucose and lactate) were evaluated (ABL 800 Flex; A. De Mori Strumenti, Milan, Italy).
At the beginning and end of the normothermic phase, perfusate samples were investigated for hepatocyte integrity (alanine-aminotransferase, ALT; aspartate-aminotransferase, AST; Lactate dehydrogenase, LDH), cholangiocellular damage (ALP, GGT), and blood count.
The concentration of total HA in outflow perfusate was determined by ELISA using a commercially available kit (sensitivity, 0.068 ng/mL; low standard, 0.625 ng/mL; R&D Systems, Minneapolis, MN, USA). The procedure was validated in preliminary experiments by using HA preparations with a known concentration and known average molecular mass (Select-HA HiLadder and LoLadder; Hyalose, Oklahoma City, OK, USA) \[[@B21-jcm-08-01918],[@B22-jcm-08-01918],[@B23-jcm-08-01918],[@B24-jcm-08-01918]\].
Sodium Citrate concentration was measured by enzymatic assay using spectrophotometry (Citrate Assay Kit, Sigma-Aldrich, Saint Louis, USA). Millipore Amicon Ultra 0.5 mL 20 KDa were used to remove proteins. Thereafter, deproteinized samples were diluted 1:2 with citrate assay buffer and centrifugated at 14,000× *g*.
2.9. Tissue Analysis {#sec2dot9-jcm-08-01918}
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Tissue samples were taken at the end of NMP in DMEM and BLOOD groups, at the end of cold storage in SCS group, and soon after laparotomy in group Native. Tissue samples (*n* = 8) were collected from the right median lobe: 1 sample was used for wet-to-dry ratio (W/D), 1 formalin fixed. Liver grafts were weighted at the end of static cold storage and the end of NMP.
2.10. Wet-to-Dry Ratio {#sec2dot10-jcm-08-01918}
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A biopsy was weighed with an analytical balance, dried in an oven at 50 °C for 48 h, and then weighted \[[@B25-jcm-08-01918]\]. Wet-to-dry ratio (W/D) was calculated and used as an index of edema. Livers procured from native and cold ischemia group were used as controls.
2.11. ATP Content Assessment {#sec2dot11-jcm-08-01918}
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Liver samples were homogenized in trichloroacetic acid (MilliporeSigma, Frankfurter, Germany), then subjected to 10 min of centrifugation at maximum speed at 4 °C. Supernatants were diluted 1:30 using 0.1 M Tris-acetate, pH 7.75 (MilliporeSigma, Frankfurter, Germany). Next, 10 mL of each sample were dispensed in a blank 96-well plate and 90 mL of luciferin/luciferase reagent were added (Enliten ATP Assay System; Promega, Madison, WI, USA). Bioluminescent signals were immediately detected with a luminometer (Glomax Luminometer; Promega, Madison, WI, USA). ATP concentration was calculated by using a standard curve that ranged from 10,211 to 1025 M (rATP 10 mM; Promega, Madison, WI, USA). Results were expressed as concentration/wet tissue weight.
2.12. Histology {#sec2dot12-jcm-08-01918}
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Liver samples were fixed in 4% formalin. Formalin-fixed-paraffin-embedded samples were stained with hematoxylin-eosin, Masson's trichrome, Periodic Acid Schiff (PAS) and reticulin histochemical staining, and CD31 immunohistochemical staining. Thirty random fields per slide were investigated to determine the necrosis area.
To evaluate liver tissue integrity, the histological samples were scored according to Brockman and colleagues \[[@B26-jcm-08-01918]\] who demonstrated concordance among NMP results, histopathological analyses, and liver viability.
2.13. Evaluation of Liver Metabolism {#sec2dot13-jcm-08-01918}
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The above-mentioned parameters and trends (glucose, lactates, potassium, and sodium citrate) were included in the liver metabolism evaluation. Citrate is metabolized by hepatocyte in the perivenular zone of hepatic lobule and its eventual decrease during the NMP procedure reflects active metabolism \[[@B