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Preservation of the liver for transplantation: Machine perfusion-based strategies for extendedpreservation and recovery

Bruinsma, B.G.

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Citation for published version (APA):Bruinsma, B. G. (2015). Preservation of the liver for transplantation: Machine perfusion-based strategies forextended preservation and recovery.

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Download date: 24 Mar 2021

CHAPTER 4

Supercooling enables long-term transplantation survival following 4 days of liver preservation

Nature Medicine; 20(7), 790-793 (2014)

Tim A. Berendsen, Bote G. Bruinsma, Catheleyne F. Puts, Nima Saeidi O. Berk Usta, Basak E. Uygun, Maria-Louisa Izamis, Mehmet Toner

Martin L. Yarmush, Korkut Uygun

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ABSTRACT

The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.

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INTRODUCTION

With over 120,000 patients waiting to receive a donor organ in the United States today, the field of transplantation is facing a serious donor shortage crisis1. Optimizing organ preservation to improve transplantation outcome has the potential to increase the availability of organs. The introduction of the University of Wisconsin (UW) solution by Belzer and Southard in the late 1980s2 represented a pivotal breakthrough in hypothermic preservation (HP) of solid organs. It substantially extended the viable preservation time3, which led to the first intercontinental kidney transplant and provided a major thrust that led to the current success of organ transplantation. To this day, donor livers are preserved in ice-cold UW solution, which offers viable preservation up to 12 h. Extension of this storage time to a hypothetical 24 h would allow larger geographic organ-sharing regions, reduce pressure on procedural logistics and optimize recipi-ent preparation. Together, such advances could contribute toward worldwide liver sharing with better outcomes, which would greatly reduce the donor shortage4. Cryopreservation has been successful in numerous cell types and some simple tissues5; however, its success for long-term storage of vascularized solid organs remains elusive owing to the adverse effects of extreme temperatures and the injurious processes necessary to reach them6,7. Experimental endeavors to achieve viable long-term preservation of whole organs at subzero temperatures range from organ freezing and vitrification as low as −196 °C4,8,9 to supercooling (subzero nonfreezing) at 0 °C to −5 °C10-16 but have yet to yield long-term survival after transplantation. Challenges in subzero preservation of liver tissue are attributable to the delicate hepatic anatomy comprising multiple cell types with variable preservation properties and functions; for instance, frozen rat livers have been transplanted with apparently healthy hepatocytes and a functioning biliary system but ultimately failed owing to endothelial cell detachment and microvascular dysfunction17. Machine perfusion, which provides organ support through an extracorporeal artificial circulation, is an alternative technique that has been shown to be advantageous over conventional hypothermic storage and has since been implemented into routine clinical practice for kidney preservation18,19. Although this method could hypothetically extend viable preservation times, its use for long-term preservation is impractical, and most studies focus on short-term preservation or recovery of warm ischemic or otherwise injured organs that would normally have been discarded19-21. In this report we present a method for extended liver storage that combines supercooling and machine perfusion. Because HP primarily works through deceleration of cellular metabolism at lowered temperatures, our central hypothesis was that supercooling would enable further metabolic reduction and extension of the viable preservation time. However, supercooling poses several challenges: (i) ice formation can occur during subzero storage and needs to be avoided; (ii) extended storage at low temperatures, along with the rewarming process, can result in irreversible plasma membrane injuries22,23 and the eventual osmotic stress that follows; and (iii) cold temperatures and subsequent rewarming increase the susceptibility of cells to oxidative damage24. This particularly affects the hepatic sinusoid, which is the functional unit of the liver that directly interacts with the exterior milieu25. The sinusoidal endothelial cells are extremely

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sensitive to hypothermic injury, which leads to detrimental microcirculatory dysfunction26,27. We developed a protocol and preservation solutions that address these issues and aim to dramatically extend liver preservation duration, as described below. For HP, the UW preservation solution has been a success28. For the purposes of supercooling, we sought to supplement the solution to reduce cold-induced membrane injury and prevent freezing. As traditional cryoprotectants such as dimethyl sulfoxide and 2,3-butanediol are toxic and may lead to issues in clinical translation, we avoided their use and employed 35-kDa polyethylene glycol (PEG), which has been shown to protect the epithelial cell membrane29,30 and has been previously used as a colloid for liver machine perfusion, as well31. Because PEG does not cross the cell membrane and is limited to extra-cellular medium, we also employed a cryoprotective additive to pre-serve the intracellular compartment. Inspired by freeze-tolerant species that produce high concentrations of glucose as a cryoprotectant32, we tested a nonmetabolizable glucose derivative (3-O-methyl-D-glucose, 3-OMG) for supercooling preservation. 3-OMG is taken up naturally by hepatocytes through glucose transporters GLUT-1 and GLUT-2 and accumulates internally. 3-OMG is nontoxic and has already been employed clinically in glucose uptake studies33. Our group has previously shown that primary hepatocytes in vitro show superior post-thaw quality when cryopreserved with 3-OMG34. To our knowledge, the study presented here is the first that has tested 3-OMG for supercooling. Finally, to counter the effects of extended ischemia, we employed machine perfusion, which has been demonstrated to alleviate hypothermic endothelial injury, reinitialize metabolic activity, replenish ATP and mechanically prime the vasculature for reperfusion19,35-37. We chose subnormothermic machine perfusion (SNMP) at 21 °C on the basis of our experience using SNMP to recover injured rat and, recently, human livers38-40. The method, as displayed in Figure 1A, entails storage of rat livers for 3 or 4 d at −6 °C, more than thrice the maximum preservation time achievable by HP, validated by orthotopic transplantation. The protocol includes the following steps. Prior to supercooling, we used SNMP with modified Williams’ E medium to load isolated rat livers with 0.2 M 3-OMG. We derived the dosage and loading characteristics of 3-OMG from in vitro experiments using isolated rat hepatocytes34. Next, we cooled the organ during SNMP to 4 °C (1 °C min-1) and flushed it with 10 ml 4 °C UW solution containing 5% 35-kDa PEG (UW-PEG). We submerged the liver in 75 ml of 4 °C UW-PEG. We then placed the liver inside a controlled-rate freezer and lowered the temperature further to −6 °C (0.1 °C min-1), initiating the supercooling phase that was maintained for 72 h (n = 6) or 96 h (n = 12). Following supercooling, we gradually increased the temperature to 4 °C. We then flushed the liver with room temperature oxygenated Williams’ E medium and subjected it to SNMP (3 h, 21 °C) while we recorded multiple viability parameters. We finally transplanted the liver orthotopically into healthy syngeneic rats. We obtained blood samples for up to 1 month and monitored the recipients for survival and clinical signs of cirrhosis for up to 3 months.

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RESULTS

Three-month recipient survival in the group that received livers supercooled for 72 h was 100%, whereas animals receiving livers exposed to an equal duration of HP died from hepatic failure within the first 2 d (Fig. 1B and Table 1). This result triples the achievable storage duration by HP, which is limited to 24 h in rat transplants. Increasing the duration of supercooling to 96 h resulted in 58% survival, which is comparable to the 50% survival achieved following 48 h of HP. Negative controls that received livers for which the SNMP loading phase or 3–OMG or PEG supplementation were individually omitted did not survive past day 6 (Table 1), whereas negative controls that received liver for which the SNMP recovery phase was omitted succumbed within 1 hour after transplantation. Despite a prolonged postsurgical recovery period and increased postoperative cellular damage parameters (Fig. 1C), recipients of livers from the 72-h and 96-h supercooling groups thrived for 3 months after transplantation without any signs of organ failure. Postoperative blood levels of albumin, bilirubin, alkaline phosphatase and blood urea normalized within 1 month after surgery, and coagulation times were normal in all animals

Figure 1. Transplantation of supercooled livers. (A) Schematic temperature (T) profile of the supercooling protocol (B) 3–month survival after transplantation of selected groups of grafts; 96–h supercooling (SC) (n=12), 72-h supercooling (n=6), 96-h hypothermic preservation (HP) (n=4), 72-h HP (n=4). Error bars, mean ± s.e.m. (C) Post-transplantation trends in transaminase output of 96-h SC (n=12) and 72-h SC (n=6) liver recipients compared to fresh liver recipients (n=6) (shown are all recipients including those that did not survive past day 1).

A

B C

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(data not shown). There were no signs (either histological, hematological or morphological) of postoperative cholangiopathy up to 3 months after surgery in any of the animals except one (from the 72-h supercooling group), which developed a large biloma 18-20 d after surgery, probably owing to blockage of the bile duct stent.

SNMP recovery phase

SNMP enables real-time evaluation of metabolic and vascular parameters. Tissue ATP levels, which dropped to approximately 10% (51.5 ± 5.8 pmol per mg protein (mean ± s.e.m.)) of fresh levels (457.2 ± 46.6 pmol per mg protein) after 96 h of supercooling, were replenished to approximately 50% of fresh levels after recovery SNMP (197.5 ± 28.4 pmol per mg protein). Although supercooled livers produced less bile than fresh livers during SNMP recovery (Fig. 2A), their bile production was substantial compared to that of livers that underwent an equal duration of HP, in which bile production was minimal. Additionally, within the 96-h supercooling group, we observed a significant difference in bile production between livers that went to recipients that survived 30 d after transplantation and those that did not (P = 0.0017, Fig. 2A). Increased hepatic resistance during SNMP recovery also correlated strongly with transplant survival in the 96-h supercooling group (P < 0.0001, Fig. 2B), suggesting that both bile production and a mean resistance <15 cm H2O min ml−1 during SNMP recovery could be predictive of liver viability. Aminotransferase output during SNMP recovery was higher than that from fresh livers in both the 72-h and 96-h supercooling groups (Fig. 2C). Within the 96-h supercooling group, we observed a significantly higher oxygen uptake in livers that went to surviving rats than in those

Table 1. Experimental groups, positive controls and negative controls used to verify the individual contributions of components of the supercooling protocol

Protocol steps n Loading phase Static preservation Recovery Survival

72-h SC 6 60-min SNMP + 3–OMG SC (–6 ºC UW–PEG solution) 180-min SNMP 100%

96-h SC 12 60-min SNMP + 3–OMG SC (–6 ºC UW–PEG solution) 180-min SNMP 58%

Fresh liver transplantation 6 - - - 100%

24-h HP 6 24 h, UW solution (4 ºC) - 100%

48-h HP 4 - 48 h, UW solution (4 ºC) - 50%

72-h HP 4 - 72 h, UW solution (4 ºC) - 0%

96-h HP 4 - 96 h, UW solution (4 ºC) - 0%

Control for recovery phase 4 60-min SNMP + 3–OMG SC (–6 ºC UW–PEG; 96h) - 0%

Control for recovery + temperature 4 60-min SNMP + 3–OMG SC (–6 ºC UW–PEG; 96h) - 0%

Control for temperature 6 60-min SNMP + 3–OMG SC (–6 ºC UW–PEG; 96h) 180-min SNMP 0%

Control for loading phase 6 SC (–6 ºC UW–PEG; 96h) 180-min SNMP 0%

Control for PEG 6 60-min SNMP + 3–OMG SC (–6 ºC UW; 96h) 180-min SNMP 0%

Control for 3–OMG 6 60-min SNMP SC (–6 ºC UW–PEG; 96h) 180-min SNMP 0%

SC, supercooling; HP, hypothermic preservation; SNMP, subnormothermic machine perfusion; 3–OMG, 3–O–methyl–D–glucose; PEG, polyethylene glycol.

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Figure 2. Subnormothermic machine perfusion recovery and histology. (A) Bile production per gram of liver during the SNMP recovery phase for 96-h SC (n = 12), 72-h SC (n = 6), 96-h HP (n = 4), 72-h HP (n = 4) and fresh control (n = 6) groups. 96-h SC survivors (n = 7) and nonsurvivors (n = 5) are represented separately for the 96-h SC group. *P < 0.01, survivors versus nonsurvivors, unpaired Student’s t-test. (B) Hepatic resistance during SNMP recovery, showing significantly higher resistance in livers that resulted in nonsurvival (*P < 0.0001, two-way analysis of variance (ANOVA)). (C) Hepatic transaminase levels in the perfusion solution during SNMP recovery. (D) Oxygen consumption during SNMP recovery. *P < 0.01, survivors versus nonsurvivors, two-way ANOVA. (E) Top, transmission electron microscopy images (1,500× initial magnification) of different stages of the supercooling protocol. Left to right, fresh liver tissue, 96-h post-supercooling liver tissue, post-SNMP recovery liver tissue and liver tissue 30 d after transplantation. The image of post-supercooling liver tissue contains a close-up (inset) of the glycogen-like structure observed in these specimens. Bottom, light microscopy images of H&E-stained tissue samples at corresponding stages of study. Error bars, mean ± s.e.m.

A B

C D

E

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that went to nonsurvivors (P < 0.0001; Fig. 2D). Liver weight did not increase significantly in all supercooled livers (2.6 ± 1.7% increase, mean ± s.e.m. P > 0.05) or during recovery SNMP (5.6 ± 2.7% increase, P > 0.05), and there were no significant differences between groups (data not shown). H&E staining and transmission electron microscopy of livers after supercooling showed normal hepatic structure in all cases (Fig. 2E). Post-supercooling samples contained intracellular accumulation of glycogen-like intracellular matter (enlarged in Fig. 2E), which may be internalized 3-OMG. In the post-transplantation specimens, we observed hepatocyte crowding and biliary hyperplasia, consistent with typical hepatocellular regeneration after rat liver transplantation.

DISCUSSION

To our knowledge, supercooling is the first preservation technique capable of rendering livers transplantable after 4 d of storage. The protocol comprises four essential components: supercooling to −6 °C, 3-OMG and 35-kDa PEG as cryoprotectants and machine perfusion for the loading of 3-OMG as well as for recovery of hepatic viability and energy stores after supercooling, before transplantation. We have shown that each of these components is individually required to achieve viable supercooling preservation in our model, as evidenced by long-term recipient survival. We show here that 100% survival is limited to 72 h of storage, as the survival drops considerably, to 58%, when the storage time is extended to 96 h. As extensive screening of different additives or variations in protocol is still ongoing, additional improvements could be achieved from future experimentation. In addition, continued investigation of the individual protocol components should provide a better understanding of the mechanisms at play. During SNMP recovery of the 96-h supercooling group, we retrospectively observed segregation of survivor and nonsurvivor recipients in terms of hepatic resistance after just 30 min of SNMP. Hence, the elevated hepatic resistance observed during the post-supercooling SNMP recovery can be interpreted as a marker for injury, which is similar to observations in nonsupercooling SNMP studies40. As a proof-of-concept small-animal study, this model has limitations and requires validation in a large-animal model. The size, robustness and preservation properties of human hepatocytes and livers differ from those of rodents, presenting translational challenges not only regarding preservation biology, but also engineering and cost. For example, this model is not suited to investigate ischemic cholangiopathy, which does not occur in rat models of liver trans-plantation. Therefore, although our work here did not reveal any indication of cholangiopathy in our supercooled rat livers, this must be examined further using a higher-order species model that utilizes clinically relevant biliary anastomosis and includes reconstruction of the hepatic artery. A second size-related issue is that in a human liver, the amount of liquid volume subject to freezing will be much larger and therefore the probability of freezing may increase; this may require additional modifications to the preservation solutions or supercooling protocol. To establish the feasibility of our method, we used fresh livers, and we demonstrated substantially preserved viability and extension of preservation time. A next major use for a superior preservation protocol would be enhancing the preservation and utilization of marginal donor organs, such as

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ischemically-injured livers donated after cardiac death. A next step will be to test and optimize the protocol described here to better preserve marginal livers and enable transplantation of currently untransplantable grafts. The achievement of long-term survival after supercooling preservation, with more than a threefold increase in the currently achievable preservation time, signifies the potential of this preservation modality. The eventual goal of extending the viable preservation time of human organs will contribute toward global organ sharing and increased organ availability.

METHODS

Brief description of the experimental protocol

The liver was procured from inbred male Lewis rats as described in detail previously41,42 (see description of liver recovery below for surgical details). Livers were subjected to subnormothermic machine perfusion (SNMP) at room temperature38,43 (see below for detailed description) through the portal vein (PV) for 60 min; the PV cuff was attached to a 18G catheter in the perfusion system. The perfusion solution (perfusate) consisted of supplemented Williams’ medium E (Sigma-Aldrich, St Louis, MO, USA) and contained the nonmetabolizable glucose 3-OMG (Sigma-Aldrich). After 60 min of loading, the temperature of the perfusate was lowered gradually (1 °C min−1) to 4 °C under continuous perfusion. Next, the liver was flushed briefly with 10 ml of UW solution containing 5% 35-kDa PEG, transferred to a sterile bag filled with the same solution and moved to a controlled-rate freezer. The temperature was lowered to −6 °C (0.1 °C min-1), and preservation was continued for up to 96 hours. The temperature was then raised to 4 °C (0.1 °C min-1), and the liver was flushed with supplemented Williams’ E medium and subjected to 3 h of recovery SNMP (see description of the supercooling procedure below for more operational details). During SNMP, measurements included liver weight, dissolved gas analysis, perfusate aspartate aminotransferase (AST) and alanine aminotransferase (ALT), bile flow and hepatic vascular resistance. The liver was then transplanted orthotopically in a syngeneic recipient animal38. We collected and analyzed blood, weighed the recipients and assessed the clinical condition for 30 d. Animals were observed for up to 3 months clinically.

Liver recovery

Inbred male Lewis rats weighing 250 to 300 g, 65-85 days old (Charles River Laboratories, Boston, MA, USA) were used for transplantation. The animals were maintained in accordance with National Research Council guidelines, and the experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (Boston, MA, USA). After dissection of the ligaments surrounding the liver, the intrahepatic inferior vena cava (IHIVC) and portal vein (PV) were elongated by dissecting their distributive veins (right renal and adrenal veins, lumbar venous plexus, gastroduodenal and splenic veins). The left diaphragmatic vein was ligated. The hepatic artery was ligated and cut. The common bile duct was cannulated for bile collection and dissected. The portal vein was cannulated, and the liver was flushed with 10 ml, 21 °C oxygenated Williams’ medium E, excised from its recess

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and transferred to the SNMP system. The portal vein and IHIVC were cuffed using a modified 16G and 14G intravenous catheter, respectively.

Subnormothermic machine perfusion

Machine perfusion took place in a circuit comprising a perfusion chamber, a peristaltic pump, a membrane oxygenator and a bubble trap. Details can be found elsewhere38,43. Temperature within the system equilibrated to room temperature at 21 °C and was not controlled otherwise. The perfusate volume was 500 ml, consisting of Williams’ Medium E supplemented with insulin (2 U l−1 Humulin; Eli Lilly & Co, Indianapolis, IN, USA), penicillin (40,000 U l−1)-streptomycin (40,000 µg l−1; Gibco/Invitrogen, Camarillo, CA, USA), L-glutamine (0.292 g l−1; Gibco/Invitrogen), hydrocortisone (10 mg l−1 Solu-Cortef; Pharmacia & Upjohn/Pfizer, New York, NY, USA) and heparin (1,000 U l−1; APP pharmaceuticals, Schaumberg, IL, USA) (during preloading, this was supplemented with 3-OMG and insulin as noted below; otherwise the loading and recovery perfusions are identical). The first 150 ml of perfusate was not recirculated before the system was closed, and a final volume of 350 ml was circulated for the remainder of the perfusion. The oxygenator was gassed with a mixture of 95% O2 and 5% CO2. Flow rate was started at 8.0 ml min−1 and adjusted according to the portal pressure, which was kept below 15 cm H2O. Aspartate aminotransferase and alanine aminotransferase were measured using an Infinity AST (GOT) and ALT liquid stable kit (Cellomics/Thermo Electron, Pittsburgh, PA, USA). Every 30 min, PV inflow and vena cava outflow were analyzed using a using a blood gas analyzer (Rapidlab 845 blood gas analyzer, Bayer). Bile was collected continually from a cannula inserted into the bile duct, which drained into a collection tube.

Supercooling procedure

Loading phase. Livers were subjected to 1 h of SNMP using oxygenated Williams’ E supplemented with 750 U l−1 insulin and 0.2 M 3-O-methyl-D-glucose. The final osmolality of the solution was 290-310 mOsmol l−1. The loading time was based partly on in vitro work with 3-OMG34, as well as preliminary trials conducted with our system (data not shown).

Supercooling phase. The SNMP system is equipped with jacketed components (perfusion chamber, oxygenator, bubble trap and perfusate reservoir) that allow circulation of antifreeze through the system on a separate circuit, enabling exact temperature regulation during machine perfusion. Liver temperature was measured using a thermocouple inserted into the suprahepatic inferior vena cava. After the 60-min 3-OMG loading phase, the temperature was reduced (1 °C min−1) to 4 °C. The liver was flushed with 10 ml ice- cold UW solution supplemented with polyethylene glycol (35-kDa PEG, 5% w/v; Sigma-Aldrich) (UW-PEG) and transferred to a sterile bag filled with 4 °C UW-PEG. To prevent thermocouple-induced ice nucleation, the temperature calibrations were performed in separate experiments; however, independent temperature monitoring of the antifreeze and UW-PEG solution was performed throughout. The liver was flushed with 10 ml UW-PEG before supercooling to ensure distribution of the preservation solution to the hepatic sinusoid. As the flush is under hypothermic conditions, washout of 3-OMG should be at a minimum, and failure to remove the SNMP perfusate from the liver would probably result in ice formation during supercooling. The

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sterile bag was sealed and immersed in ice-cold antifreeze, transferred to a controlled-rate freezer and cooled to −6 °C (0.1 °C min−1, final intrahepatic temperature between −5.5 and −6 °C); this is the lowest temperature at which ice formation could be prevented reliably over a period of 4 d in our system. The liver was preserved for 72-96 h avoiding perturbations and temperature variation.

Recovery phase. The temperature was raised to 4 °C (0.1 °C min−1); the liver was flushed with Williams’ E medium; and SNMP recovery was performed for 3 h. The recipient was prepared for surgery during perfusion recovery. Perfusion was stopped, and the liver was disconnected and flushed with 21 °C Lactated Ringer’s solution. The liver was orthotopically transplanted into a syngeneic recipient Lewis rat (250 to 300 g, 65-85 days old) as described elsewhere41. We specifically avoided the cold flush during the transplant procedure.

Post-transplantation analysis

Tail-vein blood samples were taken from transplantation recipients hourly after the procedure for 3 h, then daily for 7 d and every 5 days until day 30. The blood samples were analyzed using a Piccolo Blood Chemistry Analyzer (metabolic panel; Abaxis, Union City, CA). At 30 d post-transplantation, randomly selected livers in the 96-h SC group (n = 2) were recovered for histology and censored in the survival analysis.

Histology

Samples from dedicated livers were processed for light and transmission electron microscopy (TEM) following various stages of the supercooling protocol. For light microscopy, samples were fixed in 10% buffered formalin until further processing and stained using H&E. For EM, samples were fixed in Karnovsky’s solution.

Statistical analyses

Sample sizes were determined by power analysis based on past transplant experiments with similar variances. There was no blinding or randomization in selection of animals; however, experiments were standardized (rats were inbred Lewis rats of the same age and weight and transplantations performed at the same time of day by the same microsurgeon (T.A.B.)). Two-way (repeated measures) ANOVA was performed at α = 0.05, with post hoc Bonferroni correction for comparisons between different preservation groups over time. An unpaired student’s t-test was performed for ATP recovery and bile production between groups. A log-rank (Mantel-Cox) test was performed to compare survival curves. Normality assumption and distributions within groups were tested where appropriate.

ACKNOWLEDGMENTS

Funding from the US National Institutes of Health (grants R01EB008678, R01DK096075, R01DK084053, R00DK080942, R00DK088962 and F32 DK095558) and the Shriners Hospitals for Children is gratefully acknowledged.

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