Human livers

Ten human livers were obtained for research after being declined for transplantation nationwide and after consent was obtained from the next of kin. The local ethics committee of the Canton of Zurich approved the study protocol (2017-000412). The declined livers were procured in a standardized technique using IGL-1 for cooling and preservation. Some livers from donations after circulatory death were declined after initial hypothermic oxygenated liver machine perfusion for clinical purpose28 (Supplementary Table 1). During setup of the perfusion machine, the livers were prepared on the back table including removal of the gall bladder and cannulation of the hepatic artery, portal vein, vena cava and bile duct. Shortly before the start of liver perfusion, the preservation solution was flushed out with 2 l of cold (4 °C) Ringerfundin solution (B. Braun Melsungen) through the portal vein (1.5 l) and hepatic artery (0.5 l).

Perfusion machine and protocol for human livers

Hemodynamic control

One centrifugal pump (Thoratec CentriMag) in combination with three pinch valves (Resolution Air MPPV-8) were used to maintain the desired flow or pressure conditions in the perfusion system. Flow sensors (Sonoflow CO.56) and pressure sensors (PendoTECH Single Use Pressure Sensors) measured flow and pressure in the respective silicon tube (RAUMEDIC) lines. Thereby, the control system aimed to maintain a desired flow (for example, 1 l min−1) in the portal vein and the desired pressure conditions in the hepatic artery. To increase its resistance and provide the required flow rate ratio between the portal vein and hepatic artery, a pinch valve clamps the tube leading to the portal vein. In the hepatic artery, a pulsatile flow was applied targeting an MAP of at least 65 mmHg (for example, systolic and diastolic pressure of 80 mmHg to 50 mmHg respectively with 60 beats per minute), while limiting the mean hepatic-artery flow to a maximum of 0.6 l min−1. Pulsatile flow in the hepatic artery was realized by pulsatile operation (varying rotational speed) of the blood pump. Thereby, the rotational speed of the pump was defined in the same waveform as the desired arterial pressure pulse.

To prevent the flow in the hepatic artery from exceeding 0.6 l min−1, the system injected the vasoconstrictor Neo Synephrine HCL (phenylephrine, Ospedalia) at an upper bound of 0.55 l min−1. On the other hand, to maintain a lower limit at least 0.25 l min−1, the system injected the vasodilator Flolan (epoprostenol, GlaxoSmithKline). In both cases, this was realized by means of a proportional integral controller, defining the infusion rate on the basis of the measured hepatic-artery flow rate.

Vena cava pressure control

Inadequate blood outflow from the liver leads to a vascular congestion and an ischemic hepatocellular damage. Thus, the system controlled the vena cava pressure around the atmospheric pressure (approximately 0 mmHg).

The vena cava was connected via tube set directly to the blood reservoir. By means of the height difference between the liver and the reservoir, a suction pressure was induced and additionally controlled through a pinch valve between the vena cava and the reservoir to control a desired vena cava pressure. The pinch valve induced an additional pressure drop, allowing elevation of the pressure at the vena cava to the desired levels. If the blood pressure inside the vena cava was lower than the surrounding ambient pressure, the vessel wall collapsed (usually around 0 mmHg), closed shortly and therefore induced a certain (negative) pressure fluctuation in the VC line. This induced pressure fluctuation was utilized in the control system to target the desired vena cava pressure. Finally, this pressure fluctuation (collapse of the vena cava) was detected by the pressure sensor in the vena cava line, with the control system reacting accordingly by closing the pinch valve in the vena cava line and thereby increasing the target vena cava pressure automatically by 1 mmHg, slightly above the fluctuation point. The system continuously monitored the minimum and maximum value of the vena cava pressure of the last 30 s. If the difference between the minimum and maximum vena cava pressure in this time interval was greater than a certain threshold defined by the system (usually 10 mmHg), a fluctuation was detected. Afterward, the control system immediately increased the pressure in the vena cava line by a value of 1 mmHg, as described above. During normal operation, the system continuously searched for the fluctuation point. This was done by permanently decreasing the pressure set point for the vena cava by a rate of 1 mmHg per hour. Once fluctuation had been detected, the set point was again increased using the algorithm described above, and the system again began to search for the fluctuation pressure. This control strategy was implemented owing to the fact that the pressure distribution inside the vena cava line depended on the flow rate and therefore the vena cava pressure was also flow-rate dependent. During the perfusion duration, the total flow rate through the vena cava line always varied over time. Therefore, a steady search for the fluctuation point was implemented (Supplementary Fig. 1).

Feedback-controlled injection of insulin and glucagon

An online glucose sensor (CITSens Bio Glucose Sensor, C-CIT Sensor) was integrated into the arterial line for real-time perfusate glucose measurement. The integrated sensor continuously measured the glucose in the perfusate. The blood glucose level was kept in a range defined by the user. If the blood glucose value surpassed the desired level, the system automatically injected insulin (Actrapid, Novo Nordisk Pharma), or, if the blood glucose undershot a lower threshold, the system injected glucagon (GlucaGen, Novo Nordisk Pharma) in a closed-loop manner to maintain the perfusate glucose in a defined range. Both hormones can be kept at a basal rate as defined by the user. The injection rate of insulin was in the range of 0.02 to 4.5 IU h−1 depending on perfusate glucose level. The glucagon was injected at rates of 0.02 to 0.5 IU h−1.

Blood-gas analysis and control

By the use of an oxygenator (Medos Hilite 2400 LT) and three individual gas-flow controllers (Bronkhorst Schweiz) for oxygen, nitrogen and CO 2 , the partial oxygen pressure (pO 2 ), partial CO 2 pressure (pCO 2 ) and pH of the blood were controlled. An online blood-gas sensor (Terumo CDI 500 Shunt Sensor) continuously measured pO 2 , pCO 2 and pH. A proportional integral controller defined the oxygen concentration in the gas supply of the oxygenator to maintain pO 2 at the desired level (10–12 kPa). Another proportional integral controller defined the total gas flow rate (oxygen and nitrogen) to control the wash-out rate of pCO 2 in the perfusate. With CO 2 being present in its dissolved form as carbonic acid in the blood, the pCO 2 level could be manipulated to vary pH in a certain range. The pCO 2 was allowed to vary from 4.5 to 6.5 kPa to maintain a target pH of around 7.4. With the inverse proportionality between pCO 2 and pH, an increasing pCO 2 lead to a decrease in pH, that is, the blood became more acidic. Therefore, to reduce the pH, less pCO 2 was washed out by reducing the total gas flow rate through the oxygenator, resulting in an increase of pCO 2 . The control was implemented by means of a control cascade with the inner control loop maintaining the secondary target pCO 2 and the outer control loop maintaining the primary target pH of 7.4 by varying the pCO 2 set point in the defined range of 4.5 to 6.5 kPa. CO 2 was only supplied in the gas stream to the oxygenator before starting the liver perfusion and in the initial phase until the liver produced enough CO 2 to maintain the target pCO 2 in the blood. In the normal perfusion phase, a surplus of CO 2 is produced by the liver, requiring it to be washed out.

Physiologic oxygen saturation in portal vein

In the in vivo situation, the portal vein originates from the intestinal organs and, therefore, contains less oxygen as compared to the hepatic artery. To mimic the physiologically reduced oxygen saturation in the portal vein, venous blood from the reservoir was mixed with a portion of arterial blood leaving the oxygenator. For this purpose, the pO 2 was measured in the portal vein by means of an online blood-gas sensor (Shunt Sensor, Terumo, CDI 500) as stated above. In addition, the system continuously monitored the oxygen saturation in the vena cava (vSO 2 ) using a saturation sensor (CDI H/S Cuvette, Terumo, CDI 500). To mimic the physiological venous oxygen saturation in the portal vein, venous blood from the reservoir (blood from the vena cava with low, venous saturation) bypassed the oxygenator and was mixed with a portion of the arterial blood from the oxygenator to achieve desired saturation levels. The control was realized by means of a cascaded control. The inner control loop controlled the blood flow rate of the oxygenated blood (arterial blood) and the ‘bypass flow rate’ by means of a continuous pinch valve. The outer control loop controlled the oxygen saturation of the vena cava by varying the set point of the bypass flow rate. The goal was to keep a stable oxygenation level in the vena cava of 65% by varying the portal vein oxygen content. The total flow rate in the portal vein was maintained at 1 l min−1. An increase of flow in the bypass was automatically compensated to maintain a constant total flow rate in the portal vein.

Feedback-controlled dialysis

A dialysis loop has been integrated into the perfusion system to supply and/or remove electrolytes, bicarbonates and metabolic waste products. By defining the TMF of fluid across the dialysis filter, dialysis was further utilized to control the blood hematocrit level by adding or removing fluid from the perfusion loop. A roller pump with multiple individual channels (Ismatec Reglo ICC) controlled the inflow and outflow of dialysate to the dialysis filter (Ultraflux AV-Paed, Fresenius Medical Care), thereby allowing the system to define the TMF. The targeted TMF was defined by the user or the controller of the system. Usually, the TMF was in the range of −100 ml h−1 to 100 ml h−1. By controlling the TMF, the system can vary the amount of blood plasma in the perfusate and, thus, control the hematocrit level in perfusate. For this purpose, the control system used the continuous hematocrit measurement from the Terumo CDI 500 Cuvette sensor in the vena cava line.

Perfusate components and additives

The perfusate consisted of blood-group-matched human red blood cells (approximately 1.4 l), fresh frozen plasma (approximately 0.8 l) and 1 unit of platelets from the human blood bank. A heparin bolus (5000 U) was given at the start of perfusion. Albumin was added until it reached reference range (>2.0 g l−1). As soon as the tube set was primed with the perfusate, the dialysate (multiBIC, Fresenius Medical Care) flow rate to and from the dialysis filter was set at a rate of 1,000–2,000 ml h−1 to correct electrolytes and pH before connection of the liver to the perfusion system. The liver was connected to the system when sodium was in physiologic range and pH was around 7.2. Bicarbonates (sodium hydrogen carbonate 8.4%, B. Braun Melsungen) were also used to correct pH at dialysis start if pH was below 7.0. During liver perfusion, the dialysate inflow rate to the dialysis filter was typically set to 200 ml h−1. Dialysis outflow (leaving dialysis filter) rate was adjusted automatically by the control system depending on the measured hematocrit level. All the components of the perfusate are summarized in Supplementary Table 2.

Pig livers

The experiments were performed in accordance with the Swiss Animal Protection Law and Ordinance. Female land race pigs (around 90 kg) were obtained 1 week before surgery to allow acclimatization. The pigs were fasted overnight before the experiment with free access to water. The pigs were sedated with ketamine (Ketasol-100, 15 mg kg−1), azaperon (Stresnil, 2 mg kg−1) and atropinsulfate (Atropin 1%, 0.05 mg kg−1). General anesthesia was maintained with isoflurane (Attane, 1–2%) and propofol (Propofol Lipuro 1%, 3–5 mg kg−1 h−1). Laparotomy was performed under general anesthesia. The hepatic artery and portal vein were isolated for flow measurement with VeriQ (Medistim Germany GmbH) according to the manufacturer’s recommendation. Flow in the hepatic artery was measured distal to the splenic artery and after ligating gastric branches. Physiologic in vivo vascular flow rates in pigs for the hepatic artery and portal vein, measured at the time of organ procurement, were 0.25 ± 0.07 l min−1 (n = 5) and 1.0 ± 0.2 l min−1 (n = 5), respectively. These values served therefore as a reference for the ex vivo setting. Heparin (200 IU kg−1) was administered and the aorta was cannulated. Pig livers and blood were procured in standardized fashion22. Livers were connected to the perfusion machine after back-table preparation of the vessels (hepatic artery, portal vein, vena cava and bile duct). The circuit was primed with autologous, leukocyte-depleted pig blood (around 2.7 l) with addition of albumin (200 ml, 20% solvent), bicarbonates (20 ml), calcium gluconate (10 ml), piperacillinum–tazobactamum (2.2 g) and methylprednisolon (500 mg). The perfusion technology was developed in a step-by-step approach with the following stages as reported in the results section: glucose metabolism; minimizing hemolysis; electrolyte control; physiologic venous oxygen saturation in the portal vein; and liver movement. Pig livers were transplanted after ex vivo perfusion according to a previously established protocol22.

Sampling

Blood samples were taken before the start of perfusion, every 15 min during the first hours of perfusion and hourly for the next 5 h. Thereafter, blood sampling was performed daily. Tissue samples (liver biopsies) were collected before the start of perfusion, after 3 and 6 h of perfusion and afterward on a daily basis. Bile was sampled daily.

Measurements

Perfusate

Blood-gas analysis from arterial (hepatic artery), portal (portal vein) and caval (vena cava) lines was performed using the Radiometer ABL90 FLEX. The blood-gas analysis data were used to calibrate the glucose sensor and the online sensors of the Terumo, CDI 500 device. A Piccolo Xpress analyzer (Abaxis) was used for the quantitative determination of BUN, albumin, alanine aminotransferase (ALT), AST, total bilirubin, gamma glutamyltransferase and alkaline phosphatase using the Piccolo general chemistry 13 panel (Abaxis). Ammonia levels were determined with the PocketChem BA (PA-4140, Arkray). Factor V was measured by the clinical laboratory of the University Hospital Zurich.

Plasma from perfusate

Plasma samples were obtained from the blood-based perfusate by centrifugation using standard protocol and snap frozen in liquid nitrogen for further analysis (long-term preservation at −80 °C). Free hemoglobin was measured spectrometrically assay using Drabkin solution (D5941, Sigma). The cytokine levels in the plasma were analyzed using LEGENDplex Human Inflammation Panel (740118, Biolegend). Fifty microliters of plasma were assayed following the manufacturer’s instruction. In human livers 7 to 10, the upper measurable limit of the analytical method was taken for statistical analysis, because this value was exceeded. 8-Oxo-2′-deoxyguanosine (KA0444, Abnova) for DNA damage and cytochrome C (CSB-EL006328PI, Cusabio) for mitochondrial injury were measured with enzyme-linked immunosorbent assay according to the manufacturer’s recommendation.

Tissue

Tissue biopsies were taken at different time points and positions before and during perfusion using standard protocols, and either stored in formalin for histological analysis or snap frozen in liquid nitrogen and stored at −80 °C for further analysis.

Histology

The following staining procedures were performed on random 3-µm-thick paraffin-embedded liver sections: hematoxylin and eosin, cleaved caspase 3 (9661S, Cell Signaling) staining as an apoptosis marker, von Willebrand Factor (IR527, DAKO) for endothelial cell activation, Ki-67 staining as a proliferation marker in bile ducts (IR626, DAKO), pH3 staining as a mitosis marker (M-06-570-3KL, Milipore), CD68 staining for liver macrophages (M0876, DAKO) and cytokeratin 7 staining for bile ducts (DAKO). PAS staining was performed for qualitative analysis of glycogen content.

Frozen tissue

Total RNA was extracted from 10 mg of liver tissue using RNeasy Mini kit (Qiagen) including a DNAse I digestion step (RNase-free DNase set, Quiagen) on column. The purified RNA was transcribed to cDNA using the qScript cDNA SuperMix reagent (VWR). TaqMan gene-expression assays and an 18S rRNA internal control (TaqMan ribosomal RNA control reagents) were from PE Applied Biosystems. Sequence amplification and data analysis were performed on the ABI Prism 7500 Sequence Detector system (PE Applied Biosystems). Taqman gene-expression assays used were Ss03384604_u1 for porcine IL6, Ss03382372_u1 for porcine IL10, Ss03392385_m1 for porcine ICAM1, Hs00164932_m1 for human ICAM1 and 4308329 for 18S rRNA. The expression values were normalized to the sample at the perfusion start. Intrahepatic glycogen content was determined using the Glycogen Assay kit (MAK016, Sigma) following the manufacturer’s instructions. Total protein was extracted from tissue (10 mg) in RIPA buffer using the standard protocol. Protein quantification and immunoblots were performed using Biorad protocols and systems, and the antibodies used were phospho-Akt (S472) (9271S, Cell Signaling), α-tubulin (2125S, Cell Signaling), phospho-GSK3β (S9) (5558S, Cell Signaling) and GSK3β (12456S, Cell Signaling). Mitochondrial function was analyzed by liver ATP content using ENLITEN rLuciferase/Luciferin Reagent (FF2021, Promega). In brief, the frozen tissue (10 mg) was homogenized in 1.0 ml of ice‐cold TBS buffer and deproteinized using a TCA kit (ab204708, Abcam). One hundred microliters of diluted supernatant were assayed by addition of 50 μl of ATP monitoring reagent. Luminiscence kinetics of samples and ATP standards (Sigma) were measured using a Citation 3 imaging reader (Biotek). ATP concentrations were extrapolated from the reading of the kinetic showing better regression of the calibration curve and normalized with the protein content in the samples before deproteinization.

Bile

DRI-CHEM 4000i (Fujifilm) was used for quantitative determination of total bilirubin. Bile glucose was measured using a Radiometer ABL90 FLEX device.

Statistics

Data collection and graphs were performed with Matlab R2017a (MathWorks). The data from different experiments were bundled at the same time points. Data points were bundled to a set when at least two data points of individual experiments were available. Then, the mean and s.d. of the bundled data points was calculated. Owing to logistical reasons, the daily sampling of the various experiments could not be conducted at exactly the same times, therefore they were also bundled over a time period of a ±9 h. For comparison of two groups, two-tailed Student’s t test was used. Data were reported as mean ± s.d. P < 0.05 was considered as significant. Exact sample size (n) for each experimental group and units of measurement were provided in the text and figure captions. No assumptions or corrections were applied in statistical analysis. Further details on statistics are available online in the Reporting Summary.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.