Three livers of healthy pigs procured by standard explantation procedures, from animals used for another study (approved the Austrian federal ministry for education, science and research BMBWF-66.010/0112-V/3b/2019) by our group were used in accordance with 3Rs, the principles of laboratory animal care, and the Austrian national laws. The study was carried out in compliance with the ARRIVE guidelines.
Additionally, one human liver, rejected for transplantation due to biopsy proven steatohepatitis by all centers it was offered to, procured from a multi organ donor was used for the experiments according to the institutional ethics committee of the Medical University of Graz (30–493 ex17/18) approval.
Organ preparation and back table procedure
After procurement of the porcine organs the livers were used for MP after 3h of SCS (pig 1) or immediately after explantation (pig 2 and pig 3). For MP the A. hepatica (8F, Arterial cannula; Organ Assist, Xvivo, Groningen, Netherlands) and V. porta (25F, LiA portal cannula; Organ Assist, Xvivo, Groningen, Netherlands) are cannulated by fixation with a 2 − 0 Vicryl (Ethicon, Johnson & Johnson Medical N.V., Belgium) ligature. Furthermore, to collect the produced bile the common bile duct was cannulated with a polyurethane Nutrifit feeding tube (8F, 125cm length; Vygon, Paris, France) which was fixed by means of a 2 − 0 Vicryl (Ethicon, Johnson & Johnson Medical N.V., Belgium) ligature. Immediately prior to perfusion start, the organ was weighted and subsequently flushed with 2L of ice cold Custodiol® (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany).
The human, non-allocable liver was used immediately after delivery to the study team. Cannulation and back-table procedure was done in the same way as described above.
Sub-normothermic machine perfusion and sampling
For liver perfusion the Liver Assist® (Organ Assist, Xvivo, Groningen, Netherlands) machine is used in combination with the LiA disposable set (Organ Assist, Xvivo, Groningen, Netherlands). The 24h perfusion protocol was adapted from . The system was pre-filled with 4l of perfusion solution (Custodiol® or Custodiol MP®, Dr. Köhler Chemie, Bensheim, Germany) supplemented with Penicillin/Streptomycin (Sigma-Aldrich; Merck, Darmstadt, Germany) and Amphotericin B (Gibco; Thermo Fisher Scientific, Vienna, Austria) to a final concentration of 40,000 U/l, 0.04 mg/l and 1 mg/l respectively. Machine set-up was done according to the manufacturer’s protocol with the temperature set to 21°C and oxygenation with 100% O2 at a constant flow rate of 1l/min. After 6h of perfusion, 2L of perfusate were replaced by 2L of fresh perfusate.
Sampling (perfusate and tissue) was performed immediately prior to application of 13C-Methacetin and 1h after substrate application. Perfusate samples were shock frozen in liquid nitrogen and stored at -80°C until further use. Tissue samples were fixed in 4% Formaldehyde solution for later histological evaluation.
Non-invasive assessment of liver function by the 13CORLab Prototype
The prototype 13CORLab (ArgosMed, Karlsruhe, Germany) works on basis of isotope-selective CO2 determination enabling the detection of 12CO2 and 13CO2 separately. The machine automatically calculates 13CO2 in ppm as well as ϭ13CO2 in ‰ plotting it in real-time on the machine’s display. The latter is calculated as follows:
R depicts the ratio of concentration of 13CO2 to 12CO2 and RPDB being the Pee Dee Belemnite (RPDB = 0.0112372 )
The 13CORLab machine was connected to the air outlet of the LiverAssist® Perfusion machine (Fig. 8, yellow arrows) while all other potential air outlets were tightly closed and the oxygen supply was set to a flow rate of 1l/min. The prototype equilibrated for one hour to establish a stable baseline (BL) prior to addition of 4% 13C-Methacetin application (M1) in an organ weight-based manner. Addition of 13C Methacetin (8ml/kg of liver weight) was repeated at hours 6 (immediately after renewal of perfusate, M2), 12 (M3), and 18 (M4) immediately after routine sampling.
On basis of the ϭ13CO2 plots maximum value of ϭ13CO2, maximum delta over baseline (DOBmax), time to DOBmax as well as the area under the curve (AUC) were calculated for each 13C-Methacetin response curve (M1-M4) separately in R (version 4.1.1) in combination with R Studio (Version 1.4.1717). The area under the curves (AUC) was calculated by using the trapezoid method with the DescTools (Andri Signorell et mult. al. (2021). DescTools: Tools for descriptive statistics. R package version 0.99.43) package.
Liver specific parameters
Aspartate aminotransferase (AST), Alanine Aminotransferase (ALT), γ-glutamyltransferase (GGT), alanine phosphatase (AP) and lactate dehydrogenase (LDH) were determined from frozen perfusate samples taken immediately prior to and 1h after each 13C-Methacetin application. While the porcine parameters were determined by means of the dry-chemistry analyzer Fuji Dri-Chem NX500V (Fujifilm Europe, Düsseldorf, Germany) human parameters were analyzed on a Cobas® 8000 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) with reagents from the same manufacturer. Since the respective methods were not validate for this extraordinary sample matrix, we evaluated the analytical performance using a standard addition protocol (for details see supplemental methods).
Flavin mononucleotide (FMN) as marker for organ quality was determined based on a previously described method . Briefly, a 7-point Standard curve ranging from 10-640ng/ml was prepared by dissolving Firboflavin-5’monophospate (Sigma Aldrich, Vienna, Austria) in the desired blank perfusate. For determination of FMN levels in black 96-well plates Fluorescence was recorded at 485/520nm (Ex/Em) in duplicate by means of a Fluostar Omega (BMG Labtech, Ortenberg, Germany) plate reader. High-mobility group box 1 (HMGB-1) as marker of liver cell damage was determined by a commercially available ELISA (human: Biomatik, Delaware, USA; porcine: ByBioSource, SanDiego CA, USA) according to manufacturer’s instructions by means of a Spectrostar Omega (BMG Labtech, Ortenberg, Germany) plate reader.
Tissue preparation and evaluation
Formalin fixed paraffin embedded liver samples were prepared in a standard manner. Liver sections (1.5µm thick) were de-paraffinized and stained with hematoxylin and eosin (H&E) for evaluation by an experienced pathologist with focus on changes in inflammation, cellular necrosis and structure of liver lobes over the period of SNMP (for details refer to supplementary material).
Graphic representation of data
Graphs were drawn by means of GraphPad Prism 9 for Windows (Version 9.1.2) or R (version 4.1.1) in combination with R Studio (Version 1.4.1717) with packages ggplot2 (H. Wickham. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.), dplyr (Hadley Wickham, Romain François, Lionel Henry and Kirill Müller (2021). dplyr: A Grammar of Data Manipulation. R package version 1.0.7), ggpubr (Alboukadel Kassambara (2020). ggpubr: 'ggplot2' Based Publication Ready Plots. R package version 0.4.0), DescTools (Andri Signorell et mult. al. (2021). DescTools: Tools for descriptive statistics. R package version 0.99.43), tidyverse (Wickham et al., (2019). Welcome to the tidyverse. Journal of Open Source Software, 4(43), 1686, https://doi.org/10.21105/joss.01686)