Tissue samples of liver allografts (N = 5) subjected to NMP (Metra^®, OrganOx Limited, Oxford, United Kingdom) prior to transplantation were included in this single center experimental study between January and August 2023. The study was approved by the institutional ethics committee (Ethics Commission of the Medical University of Innsbruck, EK Nr. 1175/2018). NMP was performed in accordance with our center’s protocols as described by Cardini et al. [16, 17]. After organ acceptance and indication for NMP subjection, livers were included in the study on a rolling-basis when (i) logistics could be arranged and (ii) repetitive high-resolution respirometry measurements during the next 12 h could be ensured. At the time point of inclusion, the performance of the livers during NMP was still under investigation. Wedge biopsies (20 mg ± 2 mg wet mass) were taken during clinical NMP and divided into 4 approximately equal parts (∼5 mg each). The biopsy samples were then directly placed in ice-cold HTK solution (Custodiol^®, Dr. Franz Köhler Chemie GmbH, Bensheim, Germany) and transferred to the laboratory at 4°C on ice for further processing. The first biopsy was immediately analyzed for baseline mitochondrial respiratory function values. The further samples were stored at 4°C on ice and analyzed after 4, 8, or 12 h of HTS.

Mitochondrial respiration was assessed by HRR (O2k, Oroboros Instruments, Innsbruck, Austria) with devices equipped with two identical 0.5 mL measurement chambers at 37°C and under continuous stirring at 750 rpm [18]. Measurements were preceded by air-calibration of the devices using the mitochondrial respiration medium MiR05 (MiR05-Kit, Oroboros Instruments, Innsbruck, Austria) containing 0.5 mM EGTA, 3 mM MgCl₂ • 6 H₂O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM D-sucrose, 1 g·L⁻¹ essentially fatty acid free bovine serum albumin). Liver wedge biopsies were weighted in a 4°C environment, and subsequently homogenized in 4°C MiR05 using a PBI-Shredder O2k-Set (Oroboros Instruments, Innsbruck, Austria), to disrupt cellular structures and extract mitochondria. The crude tissue homogenate, containing freely accessible mitochondria, was adjusted to a final concentration of 1 mg wet mass·mL⁻¹, and 500 µL was transferred into the O2k chambers. Reagents were added using Hamilton glass microsyringes (Hamilton Bonaduz AG, Bonaduz, Switzerland) following pre-defined Substrate-Uncoupler-Inhibitor Titration (SUIT) protocols. All measurements were performed in technical duplicates [6]. Oxygen concentration and flux were recorded at an interval of 2 s using DatLab 7 software (Datlab 7.4, Oroboros Instruments, Innsbruck, Austria).

We applied two different SUIT protocols. SUIT(i), corresponding SUIT-025 in the online protocol library Bioblast ¹ , (Figure 1) allows for evaluation of the substrate pathway control in the OXPHOS state. After addition of ADP in saturating concentrations (5 mM), the substrates for the fatty acid oxidation-linked pathway (F _P ), malate (0.1 mM) and octanoylcarnitine (0.5 mM) were titrated, followed by cytochrome c (10 µM) for the evaluation of the integrity of the mitochondrial outer membrane. Next, substrates for the NADH-linked pathway (N _P ); pyruvate (5 mM), malate (2 mM) and glutamate (10 mM) were added. The overall capacity of convergent electron transfer pathways was determined after succinate addition (10 mM) (FNS _P ). For determination of the succinate-linked pathway alone, (S _P ) rotenone (0.5 µM) was titrated. Finally, to inhibit Complex III and determine residual oxygen consumption (ROX), antimycin A (2.5 µM) was added.

SUIT(ii), corresponding SUIT-006 in the online protocol library Bioblast ² (Figure 2) provides detailed information on the S _P -linked mitochondrial respiration. Rotenone (0.5 µM) was added for inhibition of the mitochondrial Complex I, which was followed by succinate (10 mM) titration. Next, ADP (5 mM) for evaluation of the OXPHOS capacity was added followed by cytochrome c (10 µM) for the evaluation of the integrity of the mitochondrial outer membrane. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (CCCP) was titrated stepwise (0.5 µM per step) to assess non-coupled respiration. Finally, antimycin A (2.5 µM) was added to achieve ROX.

Respiration rates were expressed as O₂ flux per wet mass tissue [pmol·O₂·s⁻¹·mg⁻¹]. In addition, respiratory capacities were normalized to an internal reference state for each measurement to determine flux control ratios (FCR) for evaluation of SUIT(i). For SUIT(ii), the coupling states LEAK (L), OXPHOS (P), OXPHOS(c) (P _c ), and Electron Transfer capacity (ET) were evaluated. Based on the above, the following control efficiencies were calculated: P-L control efficiency (1-L/P), to evaluate the efficiency of the ATP production within S _P , and cytochrome c control efficiency (1-P/P _c ) to evaluate the damage to the mitochondrial outer membrane [17, 19].

Tissue integrity and cell viability of the obtained biopsies were assessed by real-time confocal microscopy (RTCM) as previously described [20, 21]. Briefly, biopsies were incubated with the fluorescent stains Hoechst 33,342 (blue, staining all nuclei, Molecular Probes; final concentration 5 μg·mL⁻¹), propidium iodide (PI; red, staining the nuclei of dead cells, Molecular Probes; final concentration 500 nM), and wheat germ agglutinin (WGA; green, staining of cell morphology, Molecular Probes, Eugene, OR, United States; 5 μg·mL⁻¹ final concentration) at room temperature for 15 min. Image acquisition was performed with an UltraVIEW VoX spinning disk confocal system (Perkin Elmer, Wellesley, MA) connected to a Zeiss AxioObserver Z1 microscope (Zeiss, Oberkochen, Germany). Quantification relied on a semi-quantitative scoring system (0-1-2): 0—high number of viable cells, 1—an equal count of viable and dead cells, and 2—low number of viable cells [6].

Liver biopsies were fixed in 10% formalin for 24 h, then embedded in paraffin and cut in 3 µM sections. Hematoxylin and eosin (H&E) staining was performed following standard protocols to assess necrosis, inflammation, steatosis, fibrosis, and vascular changes. For scoring a modified Suzuki score was applied [6].

Shapiro-Wilk test was employed to examine the normality of the data. Continuous variables are represented as median [interquartile range (IQR)]. Categorial variables are expressed as percentage (%). To assess differences compared to baseline values (0 h sample), Friedman’s test with a post-hoc comparison to examine specific pairwise differences by the Dunn’s method was performed. Statistical analysis was performed using GraphPad Prism 10 and a p-value of <0.05 was considered statistically significant.

5 human livers undergoing NMP were included in this study. Four grafts (80%) originated from donors after brain death (DBD). The median age of the donors was 72 [54–78] years, with a median body mass index of 29 [23–33] kg·m⁻². The median donor risk index (DRI) was 2.04 [1.94–2.37], and 80% of organs were procured from ECD. One liver was found suitable for transplantation after NMP, while four livers deemed unsuitable and had to be declined. The individual donor and preservation characteristics, viability parameters and reasons for decline can be found in Table 1.

With the progression of hypothermic storage, gradual, time-dependent changes in mitochondrial respiration were observed (Figure 3).

We investigated the NADH- (N _P ), FAO- (F _P ), and succinate-linked (S _P ) oxidative phosphorylation (Figure 4; Table 2). Respiration was highest for S _P and lower for N _P and F _P . Concerning S _P , fresh and 4 h HTS measurements revealed similar respiration rates (53.59 [49.51–65.87] pmol·s⁻¹·mg wet mass⁻¹ vs. 51.01 [38.31–66.91] pmol·s⁻¹·mg wet mass⁻¹, p > 0.999), while after 8 h HTS a marked decrease in respiration was observed (43.32 [27.22–55.48] pmol·s⁻¹·mg wet mass⁻¹, p = 0.811). At 12 h, the respiration was even lower: (37.99 [19.61–47.79] pmol·s⁻¹·mg wet mass⁻¹, p = 0.06). In line with this, N _P and F _P remained stable for 4 h of HTS and declined after 8 h: N _P decreased to 3.89 [3.10–4.94] pmol·s⁻¹·mg wet mass⁻¹ after 12 h (p = 0.006). Furthermore, the flux control ratios for each pathway were calculated, to take advantage of the internal normalization relative to the maximal convergent electron pathway respiration (Figure 4; Table 2). In the present cohort, in fresh biopsies FCRs of 0.27 and 0.14 were found for F _P and N _P , respectively, which also remained constant over storage time. For S _P a FCR of 1.0 was observed for fresh biopsies and all analyzed HTS biopsies, which indicated that succinate alone is sufficient to saturate OXPHOS capacity.

The succinate-linked respiration was analyzed in greater detail as it relates to the prominent substrate pathway in the present and previous studies (Table 3; Figure 5). Measurement of LEAK respiration, representing the non-phosphorylating component of mitochondrial respiration, yielded similar results between fresh tissue biopsies and those measured after 4 h HTS (6.75 [6.69–8.50] pmol·s⁻¹·mg wet mass⁻¹ vs. 7.24 [7.03–8.09] pmol·s⁻¹·mg wet mass^-1, p > 0.999). In line with this, OXPHOS capacity, measured with saturating substrate, ADP, and inorganic phosphate concentrations remained stable for 4 h HTS (fresh 43.73 [40.69–57.71] pmol·s⁻¹·mg wet mass⁻¹ vs. 4 h HTS 44.62 [34.75–60.15] pmol·s⁻¹·mg wet mass⁻¹, p > 0.999). The same pattern was also observed for the electron transfer capacity in the non-coupled state (fresh 82.64 [78.22–93.51] pmol·s⁻¹·mg wet mass⁻¹ vs. 4 h HTS 71.09 [68.24–96.07] pmol·s⁻¹·mg wet mass⁻¹, p > 0.999). All assessed parameters showed decrease after 8 h HTS and beyond. However, significance was not reached.

The calculated mitochondrial coupling control efficiencies of the succinate-pathway did not show significant alterations during HTS up to 12 h compared to fresh biopsies (Table 4). Calculation of the P-L control efficiency allows for evaluation of the efficiency of ATP production. It was sufficiently high in fresh biopsies (0.85 [0.83–0.86]) but a trend towards lower values has been observed after storage for 12 h (0.79 [0.65–0.84], p = 0.112). Furthermore, cytochrome c control efficiency, which reflects the integrity of the mitochondrial outer membrane, revealed a slight increase at 8 h and 12 h HTS, indicating higher membrane permeability (p = 0.199 and p = 0.112) (Figure 5).

In contrast to mitochondrial respiration, tissue integrity and cell viability could be preserved for up to 12 h of HTS, as demonstrated by RTCM analysis (Figure 6). The semiquantitative RTCM score revealed no changes during the storage period (data not shown). The microscopic evaluation of H&E sections also showed no differences regarding histopathology when comparing fresh biopsies with 12 h HTS biopsies (Supplementary Figure S1). This also underlines the sensitivity of HRR in comparison to other assessment methods during cold storage.