*2.9. Tissue Analysis*

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*

A biopsy was weighed with an analytical balance, dried in an oven at 50 ◦C for 48 h, and then weighted [25]. 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*

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*

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 [26] who demonstrated concordance among NMP results, histopathological analyses, and liver viability.
