*3.1. Perfusate Composition and Hemodynamic During NMP*

Liver perfusion was started when the pH of the solution was at least 7.2. Table 1 shows the gas-analyses characteristics of the perfusate before graft connection to the NMP. Besides the differences due to the presence of red blood cells in the BLOOD group, pH, glucose, and bicarbonate concentration were statistically different at baseline gas-analyses between the groups. Baseline citrate concentration was 0.37 ± 0.05 mmol/L in the BLOOD group, while it was not detectable in the DMEM group.

During the rewarming phase, in which flow rate, temperature, and gas flow were gradually increased up to target value, there was a decrease in portal resistance in both groups (Figure 4). While in the rewarming phase portal resistance was not statistically different between the two groups (*p* = 0.105), during the normothermic phase, consistently with higher viscosity of the perfusing solution, it was higher in the BLOOD group (*p* = 0.015).


**Table 1.** Characteristics of the perfusion fluid in the two experimental groups: baseline blood-gas analyses before liver graft connection to NMP. Hb, hemoglobin; Htc, hematocrit; HbO2, hemoglobin saturation; Gluc, glucose; Lac, lactate; na, not applicable. Results are expressed as mean ± SEM.

**Figure 4.** Portal vein pressure (**A**) and resistances (**B**). PVP, portal vein pressure; PVR, portal vein resistances.

#### *3.2. Markers of Hepatocellular and Cholangiocellular Damage*

Hepatocellular integrity was evaluated during the normothermic phase (Table 2). Even if transaminases (AST) and lactate-dehydrogenase (LDH) showed a trend toward increase, this was not significant (*p* = 0.358). Furthermore, there were no differences in AST and LDH levels between the groups (Figure S3). Potassium remained stable in DMEM, while it was reabsorbed in BLOOD (*p* = 0.001). Potassium uptake ratio was also higher in the BLOOD vs. DMEM group (Figure 5A). The total amount of bile collected was higher in BLOOD group (Figure 5B). Hyaluronan decreased during the perfusion (*p* = 0.002) without any differences between the groups (*p* = 0.331).

#### *3.3. Evaluation of Liver Tissue Integrity*

W/D ratio was not different between DMEM and BLOOD groups (DMEM 3.117 ± 0.136 vs. BLOOD 2.995 ±. 0.85, *p* = 0.208). Compared to SCS group (W/D 2.872 ± 0.128), parenchymal edema was increased in DMEM (vs SCS, *p* = 0.029), while W/D ratio was not statistically different in BLOOD (vs. SCS, *p* = 0.275). Liver histological architecture was preserved in all study groups (Figure 6). Both DMEM and BLOOD showed normal histology and the grade of post-reperfusion injury was not different between the study groups (Figure 5D). As expected, red blood cells were identified within sinusoids and vessels of BLOOD group specimens. Histopathological evaluation showed no sinusoidal obstructions, hemorrhages, or endothelial damage. PAS reaction on tissue samples identified a uniform distribution of glycogen in all hepatic zones of the lobule in BLOOD, while large areas of glycogen consumption were identified in the periportal spaces of DMEM.

**Table 2.** Main metabolic and biological characteristics of normothermic machine perfusion during normothermic phase in the two study groups. \* compared to SCS group (2.872 ± 0.128) *p* = 0.029 in DMEM and *p* = 0.275 in BLOOD. ˆ compared to SCS group (985.9 ± 102.6 pmol/mg) *p* = 0.001 in DMEM and *p* = 0.012 in BLOOD. Results are expressed as mean ± SEM.


**Figure 5.** During ex-situ normothermic perfusion BLOOD group showed an increased potassium uptake ratio (\* *p* < 0.05 DMEM vs. BLOOD) (**A**) and an increased bile production (\* *p* < 0.05 DMEM vs. BLOOD) (**B**). These data suggest a more prompt and improved graft functional recovery, supported by the improved energetic pool at the end of NMP (*p* < 0.05, § vs. native, # vs. SCS, \* vs. BLOOD) (**C**). NMP preserved parenchymal architecture in both experimental groups. In BLOOD group, the use of human red blood cells did not cause sinusoidal or parenchymal damage, consistently no trapped human red blood cells were found in sinusoids (**D**). Data are shown as mean ±SEM.

**Figure 6.** Histological examination of liver biopsies. Liver histological architecture was preserved in the four study groups ((**A**) Native (untreated/control) group; (**B**) SCS group; (**C**) DMEM group; (**D**) BLOOD group). As expected, red blood cells (\*) can be found in Native and BLOOD group due to the presence of whole blood and blood based perfusate, respectively. Consistently, there were no hemorrhages or sinusoidal obstruction in BLOOD compared to the other groups. Sinusoidal architecture was preserved irrespective of the type of perfusion, as well as the rate of hepatocytes ploidy (6.2 ± 1.2/HPF). Hematoxylin and Eosin staining, 100× original magnification.
