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. 2015 Feb;15(2):381-94.
doi: 10.1111/ajt.12991.

Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions

P Fontes, R Lopez, A van der Plaats, Y Vodovotz, M Minervini, V Scott, K Soltys, S Shiva, S Paranjpe, D Sadowsky, D Barclay, R Zamora, D Stolz, A Demetris, G Michalopoulos, J W Marsh

Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions

P Fontes et al. Am J Transplant. 2015 Feb.

Abstract

We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions(21 °C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n=6) was compared to cold static preservation (CSP; n=6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100%vs. 33%; p<0.05); (2) superior graft function (p<0.05);(3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2/g/h vs. 0.096 mL O2/g/h) during MP; and (4) significantly greater bile production (MP=378.5 ± 179.7; CS=151.6 ± 116.85). MP downregulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21 °C with the HBOC solution significantly improves liver preservation compared to CSP.

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Figures

Figure 1
Figure 1
Arterial blood gases (ABGs) obtained from perfusate during machine perfusion protocol. (A) The y‐axes show oxygen pressures (pO2 mmHg) on the left and oxygen saturation (sO2%) on the right (mean ± SD). The x‐axes show the perfusion time in minutes (min). The HA pO2 is represented as pO2a. The PV pO2 is represented as pO2v. The HA saturation represented as sO2a. The PV saturation is represented as sO2v. The FiO2 was constant at 60%. (B) Carbon dioxide (CO2) pressures (mmHg) overtime (minutes) in the MP group (mean ± SD). (C) pH (mean ± SD) overtime (minutes) in the MP group.
Figure 2
Figure 2
(A) Lactate concentration (mmol/L) values (mean ± SD) in the perfusate over time (minutes) during MP. (B) Urea concentration (mmol/L) values (mean ± SD) in the perfusate overtime (hours) during MP.
Figure 3
Figure 3
Methemoglobin levels during MP and after liver implantation. (A) Methemoglobin (MetHb) saturation (%) values (mean ± SD) in the perfusate over time (minutes) during MP. (B) MetHb saturation (%) values (mean ± SD) over time (hours) in the recipient's peripheral blood during liver transplantation in the MP group.
Figure 4
Figure 4
Survival curve for both groups CSP and MP during a 5‐day period. MP had a 100% survival and CSP had a 33% survival (p < 0.05). Early deaths on the CSP group were due to primary non‐function leading into irreversible liver allograft failure.
Figure 5
Figure 5
Histological analysis of ischemia reperfusion (IR). IR scores (Suzuki modified) were determined by serial analysis of inflammatory changes within the portal tracts and the hepatic lobules. (A) Comparison of average IR scores for cold static preservation (CSP) and machine perfusion (MP). The IR scores at necropsy were significantly lower in the MP group (p < 0.05). (B) Histological images hematoxylin and eosin of the liver biopsies obtained on both groups (CSP at the top and MP at the bottom) during preservation, after reperfusion and at the end‐study necropsy.
Figure 6
Figure 6
Transmission electron microscopy analysis. The thick arrows point to the mitochondria. Machine perfusion (MP) mitochondria in the left panel have normal features. Cold static preservation (CSP) mitochondria in the right panel are swollen, which is usually an indication of mitochondrial dysfunction. The thinner arrows point to the endoplasmic reticulum (ER). MP in the left panel has normal anatomical features. CSP in the right panel show signs of ER stress. The nuclei (N) are shown as they indicate that these cells are alive and not necrotic.
Figure 7
Figure 7
AST (U/I) levels in the perfusate of both groups (CSP white; MP black) during liver preservation. MP provided effective oxygenation and significantly lower (p < 0.05) scores of hepatocellular damage when compared to CSP over a period of 9 h.
Figure 8
Figure 8
Comparison of biological processes and signaling pathways for machine perfusion (MP) and cold static preservation (CSP). (A) Biological process network analysis. Differential regulation of biological process networks during liver preservation was analyzed by comparing machine perfusion (MP) to cold storage preservation (CSP) in liver tissues after allograft reperfusion (both portal and arterial) using INGENUITY®. The biological processes that were found to be significantly affected are displayed along the y‐axis. The x‐axis displays the −log of p‐value (threshold) and was calculated using Fisher's exact test right‐tailed. The most significant up‐regulation was observed in genes associated with hepatic system development and cellular proliferation. (B) Signaling pathway analysis comparing machine perfusion (MP) and CSP after the full implantation of the liver allograft. Growth factor signaling pathways that are critical for liver function and growth were generated using INGENUITY®. The x‐axis displays the −log of p‐value (threshold) and was calculated by Fisher's exact test right‐tailed. It was observed that signaling pathways that are critical for liver growth were significantly up‐regulated in MP liver samples compared to the CSP group. NF‐κB is fully activated in hepatocyte regeneration and undergoes significant up‐regulation after partial hepatectomy.
Figure 9
Figure 9
Comparison of lipid concentration in bile of MP and CSP groups performed by metabolomics' analysis (Metabolon®, Durham, NC). The MP is brown and the CSP is blue. Bile samples were sequentially obtained through a catheter previously placed in the common bile duct (external biliary drainage). (A) Lathosterol concentration (µM) performed by gas chromatography/mass spectrometry (GC/MS) in the recipient's bile for the first 72 h after liver transplantation. (B) Campesterol concentration (µM) performed by GC/MS in the recipient's bile for the first 72 h after liver transplantation. (C) Cholesterol concentration (µM) performed by GC/MS in the recipient's bile for the first 72 h after liver transplantation.
Figure 10
Figure 10
Principal component analysis (PCA) of perfusate metabolomic profile. (A) PCA suggests importance of carbohydrate metabolism and antioxidant defenses in MP livers. PCA was carried out on the metabolomic profile of perfusate at three time points (3, 6 and 9 h). Variables are ordered by the sum of their contribution to all components, with contributions to individual components represented by different colored sections of the bars. In MP livers, variables representing carbohydrate metabolism (ribulose, ribose, glycolate) and antioxidant defenses (oxidized homo‐glutathione–GSSG) are principal drivers of metabolic changes. (B) PCA suggests importance of fatty acid metabolism in CSP livers. PCA was carried out on the metabolomic profile of perfusate at three time points (3, 6 and 9 h). Variables are ordered by the sum of their contribution to all components, with contributions to individual components represented by different colored sections of the bars. In CSP livers, PCA showed ethanolamine to be the principal driver of metabolic changes, suggesting a role for fatty acid metabolism.
Figure 11
Figure 11
Comparison of the cytokine profile for machine perfusion (MP) and cold static preservation (CSP) groups obtained at 3, 6 and 9 h during liver preservation. MP suppresses TNF‐α driven pro‐inflammatory cytokines secreted by T cells and NKT (IFN‐γ), Kupffer cells (IL‐1b) and hepatocytes (IL‐12/IL‐23) when compared to CSP. (A) MP suppresses IFN‐α secretion. (B) MP suppresses TNF‐α secretion. (C) MP suppresses IFN‐γ secretion. (D) MP suppresses IL‐4 secretion. (E) MP suppresses IL‐1β secretion. (F) MP suppresses IL‐12/IL‐23 secretion.
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