Development of a Normothermic Extracorporeal Liver Perfusion System Toward Improving Viability and Function of Human Extended Criteria Donor Livers

Transcription

1 ORIGINAL ARTICLE Development of a Normothermic Extracorporeal Liver Perfusion System Toward Improving Viability and Function of Human Extended Criteria Donor Livers Babak Banan, 1 Rao Watson, 2,3 Min Xu, 1 Yiing Lin, 1 and William Chapman 1 Departments of 1 Surgery, 2 Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; and 3 Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI Donor organ shortages have led to an increased interest in finding new approaches to recover organs from extended criteria donors (ECD). Normothermic extracorporeal liver perfusion (NELP) has been proposed as a superior preservation method to reduce ischemia/reperfusion injury (IRI), precondition suboptimal grafts, and treat ECD livers so that they can be successfully used for transplantation. The aim of this study was to investigate the beneficial effects of a modified NELP circuit on discarded human livers. Seven human livers that were rejected for transplantation were placed on a modified NELP circuit for 8 hours. Perfusate samples and needle core biopsies were obtained at hourly intervals. A defatting solution that contained exendin-4 (50 nm) and L-carnitine (10 mm) was added to the perfusate for 2 steatotic livers. NELP provided normal temperature, electrolytes, and ph and glucose levels in the perfusate along with physiological vascular flows and pressures. Functional, biochemical, and microscopic evaluation revealed no additional injuries to the grafts during NELP with an improved oxygen extraction ratio (>0.5) and stabilized markers of hepatic injury. All livers synthesized adequate amounts of bile and coagulation factors. We also demonstrated a mild reduction (10%) of macroglobular steatosis with the use of the defatting solution. Histology demonstrated normal parenchymal architecture and a minimal to complete lack of IRI at the end of NELP. In conclusion, a modified NELP circuit preserved hepatocyte architecture, recovered synthetic functions, and hepatobiliary parameters of ECD livers without additional injuries to the grafts. This approach has the potential to increase the donor pool for clinical transplantation. Liver Transplantation AASLD. Received November 2, 2015; accepted March 21, Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; C, oxygen content; CaO 2, arterial oxygen content; CIT, cold ischemia time; CvO 2, venous oxygen content; DBD, donation after brain death; DCD, donation after cardiac death; ECD, extended criteria donor; ELISA, enzyme-linked immunosorbent assay; EXP, experiment; HB, hemoglobin; H & E, hematoxylin-eosin; HMP, hypothermic machine perfusion; HTK, histidine tryptophan ketoglutarate; INR, international normalized ratio; IRI, ischemia/reperfusion injury; LDL, low-density lipoprotein; LR, lactated Ringer s; K, potassium; NA, not applicable; NELP, normothermic extracorporeal liver perfusion; NS, normal saline; PCO 2, partial pressure of carbon dioxide; PO 2, partial pressure of oxygen; PT, portal tract; RBC, red blood cell; Sa, oxygen saturation; SNMP, subnormothermic machine perfusion; TG, triglyceride; TPN, total parenteral nutrition; WIT, warm ischemia time. This project was funded by the Barnes Jewish Hospital Foundation Project Award, Transplant Research Support. The major limiting factor of using extended criteria donor (ECD) grafts is their greater susceptibility to injury during cold preservation and lower tolerance for ischemia/reperfusion injury (IRI) upon reperfusion. Therefore, ECD grafts have been associated with a higher risk of posttransplant complications such as primary graft nonfunction and chronic cholangiopathy. (1-4) Machine perfusion has been investigated both as a preservation method to reduce IRI in ECD livers and as a method to assess the viability and function of suboptimal grafts prior to transplantation. (5-7) Currently, there are 3 variants of liver machine perfusion that are distinguished by the perfusate temperature: hypothermic, subnormothermic, and normothermic. ORIGINAL ARTICLE 979

2 LIVER TRANSPLANTATION, July 2016 Although studies have shown the superiority of oxygenated perfusion over standard cold storage, each of these variants has its advantages and disadvantages. In hypothermic machine perfusion (HMP, perfusate temperature of 08C-48C), the substantial reduction in hepatocyte metabolism allows organ perfusion with preservation solutions such as histidine tryptophan ketoglutarate (HTK) or University of Wisconsin that do not have oxygen carriers. Animal liver transplantation models have successfully used HMP with donation after cardiac death (DCD) livers. (8-11) The major drawback of HMP is that prolonged perfusion during hypothermia may injure the sinusoidal endothelial cells due to increased vascular resistance and imposed shear stress, (12,13) so only short, 1-3 hour perfusion durations are feasible. In addition, HMP seems to offer reduced protection with marginal grafts. (14,15) This approach also does not permit the use of preconditioning strategies and assessment of physiological functions such as bile production. Another approach is the use of subnormothermic machine perfusion (SNMP) with perfusate temperatures at 208C-308C, which is somewhat simpler to perform than normothermic extracorporeal liver perfusion (NELP) from a technical standpoint. (16,17) Importantly, by maintaining 25% of metabolic activity, SNMP allows functional assessment of the liver. (18) Although SNMP has had promising outcomes with DCD liver transplants in animal models, (19) with a considerable reduction in the metabolism rate of the hepatocytes, it may preclude the use of preconditioning modalities such as defatting. NELP, with perfusate temperatures at 358C-378C, maintains full metabolic activity in the liver, potentially allowing the cellular regenerative process to begin to recover from ischemic Conflicts: Dr. Chapman consults for and is on the speakers bureau of Novartis. Address reprint requests to William Chapman, M.D., F.A.C.S., Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8109, St. Louis, MO Telephone: ; FAX: ; chapmanwi@wudosis.wustl.edu Additional supporting information may be found in the online version of this article. Copyright VC 2016 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /lt Potential conflict of interest: Nothing to report. damage. (20) The preconditioning of marginal organs such as with pharmacological defatting agents is another potential benefit of NELP. The technical challenges with maintaining full metabolic function on NELP include the need for an oxygen carrier, the accumulation of toxic waste products, and the need for supplementation of micronutrients. We investigate the use of a modified NELP system to perfuse human livers not suitable for transplantation for 8 hours. We demonstrate that NELP can improve the physiological and functional parameters of discarded human livers. Patients and Methods DONOR LIVERS The study protocol was reviewed and received approval by the institutional review board (IRB) committee of the Washington University and the Barnes Jewish Hospital transplant team. Between June 2014 and March 2015, 7 human livers were procured and declined for transplantation by the liver transplant team at Barnes Jewish Hospital in St. Louis, MO (Table 1). In all cases, the donors relatives provided the informed consent to use the harvested organ for research purposes. Of these, 2 were procured from a DCD donor and 5 were obtained from donation after brain death (DBD) donors. Livers were procured using a standard surgical technique. In brief, after midline laparotomy, the infrarenal aorta and the splenic vein were exposed, dissected, and cannulated with organ flush catheters; 30,000 units of heparin were administered to the donor, and upon cross-clamping of the supraceliac aorta, the abdominal organs were cooled down in situ by packing with ice, and the organs were flushed with HTK solution through the infrarenal aorta (5 L) and the splenic vein (5 L). After dissecting all attachments, liver allografts were removed from the surgical field and placed into an organ preservation bag containing cold HTK solution. An additional 1 L of cold HTK flush was performed through the portal vein. The gallbladder was incised, drained, and flushed with the cold HTK solution. Liver allografts were then stored at 48C until placed on the NELP circuit. NORMOTHERMIC EX VIVO LIVER PERFUSION All hepatic artery branches were tied off, and the patency of all vessels was checked with distal clamping of the vessels and proximal flushing with an 980 ORIGINAL ARTICLE

3 LIVER TRANSPLANTATION, Vol. 22, No. 7, 2016 TABLE 1. Characteristics of the Discarded Human Donor Livers Used for NELP Experiments. Donor Characteristics EXP001 EXP002 EXP003 EXP004 EXP005 EXP006 EXP007 DCD/DBD DCD DBD DCD DBD DBD DBD DBD Age, year Sex Male Female Male Female Male Male Male BMI, kg/m Cause of death Reason for rejection for transplantation Anoxia, drug intoxication Declined for social issues of donor Stroke Macrovesicular steatosis and mild chronic portal inflammation Anoxia, cardiovascular Rapid DCD, liver could not be placed because of timing issues Anoxia, cardiovascular Elevated transaminases and biopsy showed moderate chronic inflammation Head trauma Biopsy showed alcoholic steatohepatitis Anoxia, cardiovascular Macrovesicular steatosis and elevated transaminases Stroke Old age and poor graft quality WIT, minutes CIT, hours Preservation HTK HTK HTK HTK HTK HTK HTK solution NELP duration, hours Baseline <10% 30% <10% 30% >30% 25% <10% histological grade of steatosis (at procurement site) AST, U/L ALT, U/L olive-tipped catheter. The portal vein, hepatic artery, and common bile duct were then cannulated, and the cystic duct was ligated to prevent bile leakage during NELP. The gallbladder was then flushed with 100 ml of HTK solution to remove residual bile. The livers were flushed with normal saline (NS; 2 L from the portal vein and 1 L from the hepatic vein) prior to placement on NELP. The perfusion machine (Fig. 1) was a recirculating closed system that consisted of the following: 1 centrifugal pump (CBBP50 Carmeda pediatric Bio-Pump; Medtronic Inc., Minneapolis, MN), 1 membrane oxygenator/heat exchanger (CB3381 Carmeda pediatric Minimax Plus; Medtronic Inc.), 1 dialysis machine (Fresenius 2008K, Fresenius Inc, Concord, CA), 1 trioptic measurement cell (Medtronic Inc.) for real-time monitoring of oxygen saturation and hematocrit, 2 flow probes (Pediatric Bio-Probe; Medtronic Inc.), 2 pressure monitoring probes, and an organ chamber. The perfusate temperature was constantly regulated (at 378C) and monitored through 3 different ports: integrated thermometer on the thermoelectric water pump (Polystat[Cole-ParmerInc.],VernonHills,IL),the dialysis machine, and the integrated thermometer port on the oxygenator. The circuit was primed with 500 ml of NS and 10,000 U of heparin. Discarded (expired or near expiration) human red blood cells (RBCs) were obtained from the Barnes Jewish Hospital blood bank (the blood donors signed the consent form and gave the permission for using their blood for research purposes in case of discarding donated blood). The blood was type-matched to that of the organ donor, and 2-3 packs were added to the circuit. The perfusate was supplemented with the following: 1000 U/hour of heparin, 7 ml/hour of total parenteral nutrition (TPN; Clinimix E4.25/5, Baxter Inc., Deerfield, IL), 20 IU/hour of regular insulin, 10 lg/ hour of prostacyclin (GlaxoSmithKline Inc., London, UK), and 7 ml/hour of 1% taurocholic acid (Sigma Aldrich, MO). The NELP circuit was primed and run for minutes to allow the dialyzer to stabilize the perfusate electrolytes levels and ph. The liver was then connected to the NELP circuit and covered with a plastic sheet to avoid evaporation. The flow rate was maintained at L/minute for the hepatic artery and L/minute for the portal vein to achieve constant arterial pressures of mm Hg and portal pressures of 9 6 1mmHg.In the case of prolonged cold ischemia time, the livers were gradually rewarmed over 30 minutes on ORIGINAL ARTICLE 981

4 LIVER TRANSPLANTATION, July 2016 FIG. 1. The NELP system. The liver was placed into the liver chamber, and a centrifugal pump constantly perfused the hepatic artery and the portal vein. Both limbs were perfused with oxygenated and dialyzed blood. The dialyzate composition was as follows: sodium 137 meq/l, calcium 3 meq/l, potassium 3 meq/l, magnesium 0.75 meq/l, chloride 107 meq/l, acetic acid 4 meq/l, dextrose 100 meq/l, and bicarbonate 37 meq/l. The dialyzer pump rate was 300 ml/minute. NELP to decrease sheer stress and damage that can occur with sudden increases in temperature. The membrane oxygenator was supplemented with 95% O 2 and 5% CO 2. ASSESSMENT OF LIVER FUNCTION During NELP, samples were taken from both the arterial and venous ports and analyzed immediately for ph, partial pressure of oxygen (PO 2 ), partial pressure of carbon dioxide (PCO 2 ), HCO 3, glucose, lactate, Na 1,K 1, and Cl. The oxygen content (C) of the perfusate was calculated using Ca/vO 2 5 Hb31.393Sa/ vo 2 equation, and for calculation of the oxygen extraction ratio, Fick s equation ([CaO 2 -CvO 2 ]/CaO 2 )was used. Samples were analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Liver needle biopsies were taken at the beginning and end of the study for hematoxylin-eosin (H & E) and osmium tetroxide staining. To assess hepatocyte synthetic function, we measured the international normalized ratio (INR) in real time (CoaguChek XS system; Roche Diagnostics, Mannheim, Germany). Bile production was measured at hourly intervals. DEFATTING PROTOCOL This procedure was carried out in 2 steatotic livers. We added a mixture of 10 mm L-carnitine (Sigmatau Inc., Gaithersburg, MD) and 50 nm exendin-4 (Sigma Aldrich, St. Louis, MO) to the perfusate upon commencement of NELP. The chosen doses were based on previous reports (21-25) with effective protocols for reducing hepatocyte IRI and steatosis. Cholesterol, triglyceride (TG), and low-density lipoprotein (LDL) levels were then measured in the blood perfusate. 982 ORIGINAL ARTICLE

5 LIVER TRANSPLANTATION, Vol. 22, No. 7, 2016 Results NELP STABILIZED PH, ELECTROLYTES, VASCULAR PRESSURES, AND FLOWS AND IMPROVED HEPATIC OXYGENATION OF LIVERS PROCURED FROM ECD DONORS Human livers unsuitable for transplantation were placed upon the NELP system. The dialyzer maintained the perfusate ph and electrolyte profiles within physiologic ranges throughout the 8-hour NELP runs (Fig. 2A). The NELP system also resulted in adequate oxygenation (partial pressure of arterial oxygen, mm Hg) of the livers, leading to expected levels of carbon dioxide production (partial pressure of venous carbon dioxide, mm Hg). The oxygen extraction ratio, a marker of cellular oxygenation, was recovered within the first 10 minutes of NELP and was maintained above physiological levels in all experiments ( ; Fig. 2B). The hepatic artery and portal flows and pressures were stabilized within the first 15 minutes of NELP and maintained within normal ranges (Fig. 3); the hepatic artery and portal venous flows ranged from 0.32 to 0.52 L/minute and from 0.7 to 1.7 L/minute, respectively. The pressure ranges were mm Hg for the hepatic artery and 5-14 mm Hg for the portal vein. Liver weights before NELP ranged from 1720 to 5000 g. None of the livers had significant weight change at the end of the experiments. NELP REDUCED HEPATOCYTE INJURY AND IMPROVED SYNTHETIC FUNCTIONS OF THE GRAFTS Liver transaminases were measured to assess the liver parenchymal injury. AST and ALT levels stabilized after the fourth hour of warm perfusion without further significant elevations. Mean values were as follows: U/L at hour 4 and U/L at hour 8 for AST; and U/L at hour 4 and U/L at hour 8 for ALT. ALP values also did not rise significantly during NELP (mean values: U/L at hour 4 and U/L at hour 8; Fig. 4). These results suggest that this NELP system maintains the integrity of ECD livers. Bile production was measured as a marker of graft viability. NELP led to adequate bile synthesis, which gradually increased until hour 4 and then remained stable until hour 8. The mean bile production values were ml/hour and ml/hour at hours 4 and 8, respectively (Fig. 5A). Because the NELP circuit requires continuous administration of heparin, it was not possible to measure activated partial thromboplastin time to assess the liver synthetic function. INR was able to be used, however, and in all experiments, the INR level started high (>8) and gradually declined. The mean INR values at hours 4 and 8 on NELP were and , respectively (Fig. 5B). These results are strongly supportive that the perfused livers were recovered and produced coagulation factors. REDUCTION OF IRI AND STEATOSIS WITH NELP During this study, we were able to procure 2 steatotic livers (EXP004 and EXP005), which were perfused with a defatting solution consisting of L-carnitine and exendin-4 while on NELP. We selected L-carnitine (21) and exendin-4 (22-24) to increase the rate of fatty acid metabolism within the cytoplasm and mitochondria of hepatocytes. L-carnitine increases the rate of fatty acid transport to the mitochondria and, in fact, has been shown to be essential to b-oxidation of quarried fatty acids from the mitochondrial membrane. (26,27) It has been shown that administration of exendin-4 leads to the reduction of oxidative stress in steatotic models. (23) It protects the hepatocytes from ischemic injury by inhibiting cell death and stimulation of lipolysis. (22) We measured the levels of TG and LDL in the perfusate following NELP to evaluate the efficacy of the defatting protocol and compared the results with 1 steatotic liver that did not undergo this procedure (EXP002). Uniformly, the perfusate levels of TG and LDL rose significantly during NELP in which the defatting protocol was used; at the end of experiments, the mean TG concentration was 8.8 times higher and the mean LDL concentration was 2.6 times higher than the values at hour 0 (Fig. 5C,D). In the control liver EXP002, TG levels did not change (10 mg/dl at hours 0, 4, and 8), and LDL concentration decreased with time (5 mg/dl and 1 mg/dl at hours 0 and 8, respectively). Analysis of osmium tetroxide staining revealed a minimal reduction in the level of macroglobular steatosis in EXP004 and a 10% reduction in EXP005 (Fig. 6A-C; Table 2). EXP004 ORIGINAL ARTICLE 983

6 LIVER TRANSPLANTATION, July 2016 FIG. 2. Perfusate (A) acid-base parameters and (B) respiration properties. NELP system ensured normal levels of electrolytes and ph during warm perfusion of the liver. biopsies showed diffuse small droplet macrovesicular steatosis and 30% large droplet macrovesicular steatosis prior to NELP and minimal to no change in the amount of large and small droplet macrovesicular steatosis after 8 hours of NELP. EXP005 biopsies showed 50% small droplet macrovesicular steatosis and 80% large droplet macrovesicular steatosis prior to NELP and a 10% decrease in the amount of large droplet macrovesicular steatosis after 8 hours of NELP. No change in small droplet macrovesicular steatosis was identified. Although only 1 liver showed early signs of steatosis reduction, these preliminary results suggest that NELP and defatting agents have the potential to reduce the steatosis of livers. 984 ORIGINAL ARTICLE

7 LIVER TRANSPLANTATION, Vol. 22, No. 7, 2016 FIG. 3. Vascular hemodynamics of livers. Flows and pressures of the hepatic artery and the portal vein were constantly monitored during NELP sessions. NELP system kept the flows and vascular pressures in the physiological ranges. NELP PRESERVED HISTOLOGIC ARCHITECTURE OF HEPATOCYTES Histologic analysis revealed that following 8 hours of warm perfusion, the hepatocyte integrity was grossly normal. The livers demonstrated a minimal to complete lack of IRI (Fig. 7); biopsies from EXP003 prior to NELP showed no significant steatosis, inflammation, or fibrosis. After 8 hours on NELP, there were few histologic changes. Biopsies from EXP006 prior to NELP showed diffuse small droplet steatosis and minimal large droplet steatosis. NELP after 8 hours showed little histologic difference in steatosis from hour 0 but did show interval development of minimal IRI with minimal focal hepatocyte necrosis. Discussion In this study, we modified the NELP circuit to better support and recover human livers that had been procured from marginal donors and were considered unsuitable for transplantation. Because the aim of this study was to assess the efficacy of the NELP system and not to transplant the warm perfused livers, we performed the experiments under aseptic (not sterile) conditions. The results demonstrate that with warm perfusion, the liver maintained its synthetic functions with the adequate synthesis of bile and coagulation factors. Furthermore, NELP stabilized and reduced IRI of the livers that were considered unsuitable for transplant. In addition, preliminary results suggest that graft steatosis may be reduced with the use of defatting protocols on NELP. Because of the complexity of the normothermic perfusion systems along with the unpredictable nature of using discarded human livers for research purposes, there have been very few published studies that have tested the outcomes of warm perfusion on marginal and steatotic human livers that were not used for clinical transplantation. Therefore, there is no uniform data about a proper viability assessment and functionality markers of discarded human livers during NELP. ORIGINAL ARTICLE 985

8 LIVER TRANSPLANTATION, July 2016 FIG. 4. Hepatocyte injury markers. Concentrations of AST, ALT, and ALP in the perfusate were measured as the markers of hepatobiliary injury. NELP preserved hepatocyte architecture and stabilized markers of hepatocyte injury during the warm perfusion sessions. Historically, measuring bile production and concentration of coagulation factor V (enzyme-linked immunosorbent assay [ELISA]) have been used as markers of liver viability and functionality. (28-30) Unfortunately, bile production is a very subjective measurement, and to date, there has not been any published study about the acceptable range for satisfactory bile production in ex vivo models. Moreover, livers that do not make bile in a short period of NELP may start producing bile later in the course of warm perfusion. Also, bile production can be falsely increased by continuous supplementation of the NELP circuit with taurocholic acid. In addition, given the costs and also the duration needed for performing ELISA, it is not possible to use this assay for measuring the concentration of factor V in real time. We have previously shown that measurement of INR during NELP experiments is a reliable approach for the assessment of synthetic functions of the liver. (31,32) It is fast (results less than 2 minutes) and can be checked in real time. Despite ELISA kits, measuring INR is not costly, and more importantly, it is a less subjective and more quantitative marker than the bile production. Results presented in this communication demonstrated that all 7 ECD livers were able to start producing coagulation factors (as evident by constant reduction in INR values) after starting the warm perfusion. Therefore, we feel that this approach can be easily adapted to clinical scenarios to assess the function of the liver while it is on NELP and can be a marker for its potential clinical use. Maintaining a metabolically active liver at normothermic conditions mandates using an oxygen carrier for sufficient gas exchange. However, hemolysis of the RBCs during ex vivo perfusion remains the major obstacle that hinders long warm perfusion experiments. Hence, novel oxygen carrier solutions that efficiently deliver oxygen to the organ are certainly needed to overcome the hemolysis problem as the main hurdle during ex vivo perfusion. (33) Defatting is a new approach in the liver preservation field, which has been shown to be possible in animal models by ex vivo normothermic systems. In a 986 ORIGINAL ARTICLE

9 LIVER TRANSPLANTATION, Vol. 22, No. 7, 2016 FIG. 5. (A, B) Synthetic parameters of the liver and (C, D) defatting parameters in the perfusate. All livers produced desirable amount of bile as well as coagulation factors. Adding the defatting solution to the perfusate led to significant rise in the TG and LDL concentrations in the perfusate. preliminary study using steatotic porcine livers, Jamieson et al. showed a 50% reduction in the steatosis level after 48 hours of NELP. (34) Moreover, normothermic perfusion of steatotic livers from obese Zucker rats using a defatting cocktail led to a 50% reduction in intracellular lipid content after 3 hours of machine perfusion. (25) In our preliminary experiments, we employed 2 steatotic human livers not suitable for clinical transplantation and used a defatting protocol at the beginning of NELP. The results demonstrated a 10% reduction in the level of macroglobular steatosis in 1 liver (Table 2). Both livers did not show any pathological changes associated with IRI (Fig. 5). Although the amount of steatosis reduction was minimal, it supports previous animal studies demonstrating the potential of NELP to reduce the steatosis level with appropriate defatting agents without increasing IRI, which suggests that this approach may have clinical potential. It is likely that with a longer duration of NELP or constant supplementation of defatting solution one can expect a significant reduction in the steatosis level. However, without a standard control group in a defatting experiment, the presented data are descriptive and not conclusive. Certainly, this approach is not ready for clinical purposes yet, and future dose adjustment experiments with mechanistic approaches are needed to uncover the actual impact of this defatting solution on the liver allografts during NELP. Adding a dialysis circuit to the system can solve some of the most important challenges of the NELP system. A rise in urea and glucose levels is a major problem during warm perfusion sessions, leading to a significant increase in the perfusate osmolality. (35) Our preliminary observations (without dialyzer) using porcine livers yielded inferior results (poor bile production, high INR, and high AST and ALT) along with intractable acid-base abnormalities during NELP runs (Supporting Fig. 1). After incorporating the dialyzer, excess glucose and urea were constantly removed, and perfusate osmolality was kept in the physiological range leading to significantly better results. Because the liver metabolism rate is at the maximum during the warm perfusion experiments, besides providing sufficient oxygenation and removing produced CO 2 from the system, the allografts need to be supplemented with nutrients to prevent cellular ORIGINAL ARTICLE 987

10 LIVER TRANSPLANTATION, July 2016 FIG. 6. Histologic review of osmium tetroxide stained steatotic liver sections of (A) EXP004 and (B) EXP005 and (C) H & E stained sections of EXP002. EXP004 biopsies showed diffuse small droplet macrovesicular and 30% large droplet macrovesicular steatosis (at 2003 and 4003 magnification) prior to NELP and minimal to no change in the amount of large and small droplet macrovesicular steatosis (at 2003 and 4003 magnification) after 8 hours of NELP. EXP005 biopsies showed 50% small droplet macrovesicular steatosis and 80% large droplet macrovesicular steatosis (at 2003 and 4003 magnification) prior to NELP and a mild decrease in the amount of large droplet macrovesicular steatosis (at 2003 and 4003 magnification) after 8 hours of NELP. No change in small droplet macrovesicular steatosis is identified. EXP002 biopsies revealed minimal to no change in the level of steatosis (at 2003 and 4003 magnification), increased IRI, and hepatocyte necrosis after 8 hours of NELP. 988 ORIGINAL ARTICLE

11 LIVER TRANSPLANTATION, Vol. 22, No. 7, 2016 FIG. 6. Continued. TABLE 2. Experimental Values EXP004 EXP005 EXP002 Hour 0 Hour 8 Hour 0 Hour 8 Hour 0 Hour 8 Steatosis 30% 30% 80% 70% 10% 10% (large droplet) Steatosis 100% 100% 50% 50% 20% 10% (small droplet) Microvesicular steatosis Ballooning NA NA NA NA NA NA Satellitosis NA NA NA NA NA NA PT inflammation No No No No Mild Minimal Lobular inflammation No No No No Mild Minimal damage. Adding TPN or some kind of nutritional supplementation to the NELP circuit has been previously reported by several other groups. (28,30,36-38) However, the exact dosing of the TPN and the metabolic consequences of adding this solution during machine perfusion has yet to be investigated. Therefore, further mechanistic studies are needed to uncover the specific allograft supplemental requirements and to deeply understand the metabolic pathways changes during ex vivo perfusion experiments. In the majority of published studies of ex vivo models (referenced above), the hepatic artery and portal vein pressure ranges are and 5-12 mm Hg, respectively. Our data fit in about the same pressure ranges. Higher pressures at the first hour of machine perfusion are certainly due to graft ischemia and subsequent vasoconstriction, especially in the arterial side. In our experiments, the vascular pressures declined after 1 hour of warm perfusion. We supplemented both the hepatic artery and the portal vein with ORIGINAL ARTICLE 989

12 LIVER TRANSPLANTATION, July 2016 FIG. 7. Histologic review of H & E stained liver sections of (A) EXP003 and (B) EXP006. Biopsies from EXP003 prior to NELP showed no significant steatosis, inflammation, or fibrosis. NELP after 8 hours showed little histologic difference from hour 0 (at 2003 and 4003 magnification). Biopsies from EXP006 prior to NELP showed diffuse small droplet steatosis and minimal large droplet steatosis. NELP after 8 hours showed little histologic difference in steatosis from hour 0 but interval development of minimal IRI with minimal focal hepatocyte necrosis (at 2003 and 4003 magnification). continuous flows. Therefore, a pulsatile flow to the hepatic artery was not provided. Although, having 2 different types of flows (pulsatile and continuous) to the hepatic artery and the portal vein simulate normal liver physiology, but the necessity and efficacy of providing pulsatile flow to the hepatic artery has not been comprehensively investigated. Because we have achieved reproducible and satisfactory results in our 990 ORIGINAL ARTICLE

13 LIVER TRANSPLANTATION, Vol. 22, No. 7, 2016 previous projects (31,32) with providing continuous flow to both vascular limbs, we preferred to keep the system as simple as possible with using only 1 centrifugal pump. Nonetheless, we have designed a new system that will enable us to supply pulsatile and continuous flows by using only 1 centrifugal pump. Indeed, in future studies, we will test the efficacy of the newly designed system. Although it is accepted that the aim of NELP is to provide a physiologic environment for the liver allograft, we want to extend this aim to provide a supra physiologic environment for the compromised livers. The dialyzer enabled us to manipulate the key elements of IRI process. (39) For example, it has been shown that calcium overload in the first few minutes after reperfusion is one of the major players of the IRI cascade that leads to excessive production of reactive oxygen species and destructive damage to the mitochondria. (40-42) Therefore, we set the calcium levels at 3 meq/l (1.5 mmol/l; subnormal concentration) by the dialyzer in all NELP experiments. The dialyzer also removed excess potassium from RBC destruction and maintained the levels below 4 mmol/l (Fig. 2A). In addition, calcium and potassium overload during the back-table procedure were avoided by flushing the liver allografts with NS instead of lactated Ringer s (LR) solution. LR solution contains 4 mmol/l of potassium, 3 mmol/l of calcium, and 28 mmol/l of lactate that could potentially lead to reperfusion injuries upon connecting the sensitive ECD allografts to the NELP system. In contrast, the NS solution contains only sodium and chloride ions and does not impose the allograft to the potential adverse effects of calcium and potassium ions. Also, the NELP perfusate solution consisted of the packed RBCs diluted with the NS. Therefore, because the NS solution acted as the base for our NELP circuit, we flushed the livers with this solution and not with LR. Lactic acid accumulation can lead to free-radicalmediated injury and potentially aggravating IRI cascade. (43,44) Another benefit of adding the dialyzer to the circuit was the constant clearance of lactic acids and stabilization of ph during NELP. In all our experiments, despite a transient increase in lactate levels at the beginning of the warm perfusion, its levels decreased dramatically after minutes of NELP to below 0.5 mmol/l (data not shown). Traditionally, lactate concentration has been used as a marker of graft functionality during ex vivo perfusion, (20,28,45) but we believe that removing lactic acids from the NELP circuit and stabilizing ph without adding several doses of sodium-bicarbonate to the circuit (especially during the first 30 minutes of NELP) is a superior strategy for recovering a liver especially in longer machine perfusion durations. Clinical markers of organ functionality are not always applicable to ex vivo experiments. In fact, ex vivo models need an ex vivo approach regarding assessing the functionality of the liver allografts. For example, in the clinical setting, a constant declining glucose level is an indicator of liver failure. However, in ex vivo normothermic perfusion models, this is a good sign of liver functionality. In many studies (including ours), despite an initial immediate increase in the glucose levels upon connecting the liver to the NELP system, a constant reduction of glucose has been reported. (28,36,46) In fact, it has been shown that a constant reduction in perfusate glucose concentrations during NELP is accompanied by increased glycogen storage in the hepatocytes. (47) On the other hand, glucose decline during NELP is a good sign of liver functionality and shows that the glucose is constantly being picked up by the hepatocytes and is being stored as glycogen. Taken all together, with having biopsies, INR, and bile production, we believe that the observed glucose reduction is not an indicator of graft dysfunction. The authors would like to clarify the limitations of this protocol and explain that the presented communication is a preliminary report and lacks a clinical aspect. It was not designed as a transplantation model, and the livers were not reperfused with the recipient s blood. Therefore, at this time, we are not able to comment on the attenuation of IRI in a real postreperfusion phase when the liver allografts have been implanted into the recipient s abdomen. Future transplantation models have been designed to assess the outcomes of transplanting NELP-perfused liver allografts. Another limitation of this study is the small number of donor livers and lack of a proper control group for comparison purposes. Certainly, with higher numbers of donor livers and larger experimental groups, more informative data could be gathered, and the conclusion, discussion, and the message would have been much stronger. However, using discarded human livers for research purposes is not a straightforward and fastpaced project. As mentioned in the Patients and Methods section, we were able to perform successful NELP experiments on only 7 ECD livers in 1 year. Given the NELP system challenges from the technical standpoint and also the scarcity of ECD livers that become available for research purposes, performing ORIGINAL ARTICLE 991

14 LIVER TRANSPLANTATION, July 2016 more NELP runs and collecting more data from more livers would dramatically extend the intended duration of this study from 1 year to 2-3 years. Indeed, the majority of published studies about ex vivo perfusion of discarded human livers have reported using more or less the same number of livers. (18,28,35,36) Moreover, due to facing various challenges with using discarded human livers, in the majority of these publications, the authors have preferred to report a review of their data and to describe the outcomes of their study rather than designing a full control-versus-treatment-like project. Therefore, although we believe that this project would certainly benefit from incorporating a higher number of livers, unfortunately, logistics of organ allocation for research purposes and practical challenges of ex vivo perfusion models have limited us to do so. 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