Parameters of hepatocellular secretory function of DCD livers that were preserved by either 4 h of oxygenated HMP or SCS and subsequently reperfused for 2 <b>h by normothermic </b><i>ex vivo</i> sanguineous perfusion.

<p><i>Panel A</i>: Evolution of bile production in the HMP and SCS group. <i>Panel B and C</i>: Biliary concentration of bile salts and phospholipids, respectively. <i>Panel D</i>: Bile salt toxicity, as represented by the ratio of biliary bile salt and phospholipid concentrations. There were no statistically significant differences between the groups. <i>Panel E–F</i>: Relative mRNA expression of the main hepatocellular bile transporters BSEP (bile salt export pump; Abcb11) and MDR3 (multidrug resistance protein 3, Abcb4) after 2 h of <i>ex vivo</i> sanguineous reperfusion of DCD livers that were preserved by either 4 h of oxygenated HMP or SCS. There were no significant differences between the two groups.</p

Representative examples of histology of liver parenchyma of DCD liver grafts preserved by either 4 h of SCS or 4 h of oxygenated HMP followed by 2 h by normothermic <i>ex vivo</i> sanguineous perfusion.

<p><i>Panel A</i>: H&E staining of a central biopsy of the liver parenchyma. <i>Panel B</i>: Caspase-3 immunohistochemistry of liver parenchyma showing less intense caspase-3 staining of hepatocytes, sinusoidal endothelial cells, and Kupffer cells in the HMP group, compared to the SCS group. Brown color indicates immunopositivity. Original magnification 200x.</p

Comparison of biochemical parameters of bile duct injury and oxidative stress in DCD livers that were preserved by either 4 h of oxygenated HMP or 4 h of SCS and subsequently reperfused for 2 h by normothermic <i>ex vivo</i> sanguineous perfusion.

<p><i>Panel A–C</i>: Concentration of LDH, alkaline phosphatase, and gamma-GT in bile samples at 2 h after reperfusion. <i>Panel D</i>: Comparison of biliary concentration of TBARS, a marker for oxidative stress and lipid peroxidation in bile ducts, at 2 h after graft reperfusion. There were no significant differences between the SCS preserved and HMP preserved livers.</p

Representative examples of H&E histology of bile ducts of DCD liver grafts preserved by either 4 h of oxygenated HMP or 4 h of SCS followed by 2 h by normothermic <i>ex vivo</i> sanguineous perfusion.

<p><i>Panel A</i>: extrahepatic bile duct immediately after procurement (magnification 200x). The insert represents a higher magnification of 400x. <i>Panel B</i>: extrahepatic bile duct after preservation and reperfusion (magnification 280x). <i>Panel C</i>: Immunohistochemistry for activated caspase-3 of the same bile ducts as presented in panel B (brown color indicates immunopositivity; counterstaining with hematoxylin). Very few caspase-3 positive cells were detected in the bile duct wall stroma. Remnant biliary epithelial cells (i.e. in the peribiliary glands) were not positive for acitivated caspase-3. <i>Panel D</i>: higher magnification (400x) of extrahepatic bile ducts focusing on the peribiliary plexus. Arrows indicate peribiliary arterioles. The insert represents a higher magnification of the peribiliary arterioles (800x). Bile ducts of livers preserved by oxygenated HMP displayed significantly less signs of arteriolonecrosis, compared to livers preserved by SCS (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088521#pone-0088521-t001" target="_blank"><b>Table 1</b></a>).</p

Comparison of functional parameters of biliary epithelial cell function in DCD livers that were preserved by either 4 h of oxygenated HMP or 4 h of SCS and subsequently reperfused for 2 h by normothermic <i>ex vivo</i> sanguineous perfusion.

<p>There were no significant differences in biliary pH (<i>Panel A</i>), biliary bicarbonate (<i>Panel B</i>) and glucose concentration (<i>Panel C</i>) at 2 h after reperfusion between the two groups. Relative mRNA expression of the main cholangiocyte transporter proteins involved in biliary bicarbonate secretion, CFTR (cystic fibrosis transmembrane conductance regulator; ABC35) (<i>Panel D</i>) and AE2 (anion exchanger 2; SLC4A2) (<i>Panel E</i>) after 2 h of <i>ex vivo</i> sanguineous reperfusion of DCD livers that were preserved by either 4 h of oxygenated HMP or SCS. There were no significant differences between the two groups.</p

Biochemical Composition of Perfusion Fluid and Bile after 6 hour of <i>Ex Vivo</i> Normothermic Machine Perfusion.

<p>* To convert values for glucose to mg/dL, multiply by 18.02. To convert values for lactate to mg/dL, multiply by 9.01. To convert values for hemoglobin to g/dL, multiply by 1.650. To convert the value for bilirubin to mg/dL, divide by 17.1. To convert kPa to mmHg, multiply by 7.5.</p><p>** Peak values during 6 h of machine perfusion.</p><p>Abbreviations used: ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamate transferase; LDH, lactate dehydrogenase.</p><p>Biochemical Composition of Perfusion Fluid and Bile after 6 hour of <i>Ex Vivo</i> Normothermic Machine Perfusion.</p

Criteria to Assess Bile Production after 2.5 hours of Normothermic Machine Perfusion.

<p>Criteria are: 1) Cumulative bile production of ≥10 grams after 2.5 h and 2) a bile production of ≥4 grams in the preceding hour (1.5–2.5 h of perfusion).</p><p>Criteria to Assess Bile Production after 2.5 hours of Normothermic Machine Perfusion.</p

Changes in portal flow (panel A) and arterial flow (panel B) during <i>ex vivo</i> normothermic machine perfusion of human donor livers, using a pressure controlled device.

<p>Flow in the portal vein and hepatic artery increased rapidly during the first 30 min and flows remained stable thereafter for the entire 6 h perfusion period. There were no significant differences in portal flow and although median arterial flow was constantly lower in livers with a low bile output, compared to the high bile output group, this did not reach statistical significance.</p

Changes in hepatic energy content as reflected by hepatic ATP content.

<p>In contrast to livers with low bile output, livers in the high bile output group showed a significantly higher hepatic ATP content during the course of NMP. (AUC p = 0.04).</p

Biochemical Composition of Perfusion Fluid Used For Normothermic Machine Perfusion of Donor Livers.

<p>* To convert values for glucose to mg/dL, multiply by 18.02. To convert values for lactate to mg/dL, multiply by 9.01. To convert values for hemoglobin to g/dL, multiply by 1.650. To convert the value for bilirubin to mg/dL, divide by 17.1.</p><p>Biochemical Composition of Perfusion Fluid Used For Normothermic Machine Perfusion of Donor Livers.</p