Bile duct regeneration is hypothesized to prevent biliary strictures, a leading cause of morbidity after liver transplantation. Assessing the capacity for biliary regeneration may identify grafts as suitable for transplantation that are currently declined, but this has been unfeasible until now. This study used long-term ex situ normothermic machine perfusion (LT-NMP) to assess biliary regeneration. Human livers that were declined for transplantation were perfused at 36 °C for up to 13.5 days. Bile duct biopsies, bile, and perfusate were collected throughout perfusion, which were examined for features of injury and regeneration. Biliary regeneration was defined as new Ki-67-positive biliary epithelium following severe injury. Ten livers were perfused for a median duration of 7.5 days. Severe bile duct injury occurred in all grafts, and biliary regeneration occurred in 70% of grafts. Traditional biomarkers of biliary viability such as bile glucose improved during perfusion but this was not associated with biliary regeneration (P > .05). In contrast, the maintenance of interleukin-6 and vascular endothelial growth factor-A levels in bile was associated with biliary regeneration (P = .017 for both cytokines). This is the first study to demonstrate biliary regeneration during LT-NMP and identify a cytokine signature in bile as a novel biomarker for biliary regeneration during LT-NMP.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Recent advances in machine perfusion have revolutionised the field of transplantation by prolonging preservation, permitting evaluation of viability prior to implant and rescue of discarded organs. Long-term perfusion for days-to-weeks provides time to modify these organs prior to transplantation. By using long-term normothermic machine perfusion to facilitate liver splitting and subsequent perfusion of both partial organs, possibilities even outside the clinical arena become possible. This model remains in its infancy but in the future, could allow for detailed study of liver injury and regeneration, and ex-situ treatment strategies such as defatting, genetic modulation and stem-cell therapies. Here we provide insight into this new model for research and highlight its great potential and current limitations.

BACKGROUND:Normothermic machine perfusion (NMP) allows for the assessment and resuscitation of ex-vivo human livers prior to transplantation. Commercially available NMP systems are closed circuits that accumulate metabolic waste and cytokines over time, potentially limiting organ preservation times. Dialysis has been proposed as a method to remove waste and excess fluid from such systems. This study aimed to demonstrate the utility of integrating dialysis into a commercially available system by quantifying solute removal. METHODS:A dialysis filter was attached in parallel to a commercially available liver perfusion system. Three livers declined for transplantation were split before undergoing long-term NMP with blood using the modified system. During perfusion, dialysate flow rates were set in the range of 100-600 mL/h for short periods of time. At each flow rate, perfusate and spent dialysate samples were collected and analyzed for solute clearance. RESULTS:The addition of dialysis to a commercial NMP system removed water-soluble waste and helped regulate electrolyte concentrations. Interleukin-6 was successfully removed from the perfusate. Solute clearance was proportional to dialysate flow rate. A guide for our perfusion setup was created for the appropriate selection of dialysis flow rates and duration based on real-time perfusate composition. CONCLUSIONS:Dialysis circuits can efficiently remove waste and regulate perfusate composition, and can be easily incorporated to improve the performance of commercially available systems. Quantification of the effect of dialysis on perfusate composition enables refined dialysis control to optimize electrolyte profiles and avoid the over- or under-correction of key solutes.

BACKGROUND:Normothermic machine perfusion permits the ex vivo preservation of human livers before transplantation. Long-term perfusion for days-to-weeks provides the opportunity for enhanced pretransplant assessment and potential regeneration of organs. However, this risks microbial contamination and infection of the recipient if the organ is transplanted. An understanding of perfusate microbial contamination is required to inform infection control procedures and antimicrobial prophylaxis for this technology.METHODS:We modified a liver perfusion machine for long-term use by adding long-term oxygenators and a dialysis filter. Human livers that were not suitable for transplantation were perfused using a red-cell-based perfusate under aseptic and normothermic conditions (36 °C) with a goal of 14 d. Cephazolin was added to the perfusate for antimicrobial prophylaxis. Perfusate and bile were sampled every 72 h for microbial culture.RESULTS:Eighteen partial human livers (9 left lateral segment grafts and 9 extended right grafts) were perfused using our perfusion system. The median survival was 7.2 d. All organs surviving longer than 7 d (9/18) had negative perfusate cultures at 24 and 48 h. Half of the grafts (9/18) became culture-positive by the end of perfusion. Microbial contaminants included Gram-negative ( Pseudomonas species, Proteus mirabilis, Stenotrophomonas maltophilia ) and Gram-positive bacteria ( Staphylococcus epidermidis , Enterococcus faecalis , and Bacillus species) as well as yeast ( Candida albicans ).CONCLUSIONS:Microbial contamination of perfusate is common during long-term perfusion of human livers with both exogenous and endogenous sources. Enhanced infection control practices and review of targeted antimicrobial prophylaxis are likely to be necessary for translation into the clinical arena.

Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.

Introduction. Biliary complications are a common cause of morbidity after liver transplantation and associated with bile duct injury. To reduce injury, a bile duct flush is performed with high-viscosity preservation solution. It has been suggested that an earlier additional bile duct flush with low-viscosity preservation solution may reduce bile duct injury and biliary complications. This study aimed to investigate whether an earlier additional bile duct flush would reduce bile duct injury or biliary complications. Methods. A randomized trial was conducted using 64 liver grafts from brain dead donors. The control group received a bile duct flush with University of Wisconsin (UW) solution after donor hepatectomy. The intervention group received a bile duct flush using low-viscosity Marshall solution immediately after the onset of cold ischemia and a bile duct flush with University of Wisconsin solution after donor hepatectomy. The primary outcomes were the degree of histological bile duct injury, assessed using the bile duct injury score, and biliary complications within 24 mo of transplant. Results. Bile duct injury scores were not different between the 2 groups. Similar rates of biliary complications occurred in the intervention group (31% [n = 9]) and controls (23% [n = 8]) (P = 0.573). No difference between groups was observed for anastomotic strictures (24% versus 20%, P = 0.766) or nonanastomotic strictures (7% versus 6%, P = 1.00). Conclusions. This is the first randomized trial to investigate an additional bile duct flush using low-viscosity preservation solution during organ procurement. The findings from this study suggest that performing an earlier additional bile duct flush with Marshall solution does not prevent biliary complications and bile duct injury.

Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.

Introduction: Histological injury to the biliary tree during organ preservation leads to biliary strictures after liver transplantation. The Bile Duct Injury (BDI) score was developed to assess histological injury and identify the grafts most likely to develop biliary strictures. The BDI score evaluates the bile duct mural stroma, peribiliary vascular plexus (PVP) and deep peribiliary glands (DPGs), which were correlated with post-transplant biliary strictures. However, the BDI score has not been externally validated. The aim of this study was to verify whether the BDI score could predict biliary strictures at our transplant centre. Methods: Brain-dead donor liver grafts transplanted at a single institution from March 2015 to June 2016 were included in this analysis. Bile duct biopsies were collected immediately before transplantation and assessed for bile duct injury by two blinded pathologists. The primary outcome was the development of clinically significant biliary strictures within 24 months post-transplant. Results: Fifty-seven grafts were included in the study which included 16 biliary strictures (28%). Using the BDI score, mural stromal, PVP and DPG injury did not correlate with biliary strictures including Non-Anastomotic Strictures. Severe inflammation (>50 leucocytes per HPF) was the only histological feature inversely correlated with the primary outcome (absent in the biliary stricture group vs. 41% in the no-stricture group, p = 0.001). Conclusions: The current study highlights limitations of the histological assessment of bile duct injury. Although all grafts had bile duct injury, only inflammation was associated with biliary strictures. The BDI score was unable to predict post-transplant biliary strictures in our patient population.

Background: Split liver transplantation permits the transplant of two recipients using a single donor liver. Liver splitting can be performed using the ex-vivo technique (more convenient), or the in-situ technique (shorter cold ischaemic time). We aimed to develop a technique for liver splitting during normothermic machine perfusion which combines the advantages of both techniques and permits graft assessment prior to transplant. Methods: Human livers declined for transplantation were perfused at 36 degrees C using a modified -commercial perfusion machine. We developed a six-step method to split whole livers into left lateral segment grafts and extended right grafts. Both partial livers were then perfused on separate machines for individual assessment. Results: Using our technique, 10 whole livers were successfully split during normothermic perfusion resulting in 20 partial grafts. Apart from a single graft which failed due to a technical error, all grafts survived for 24-h after splitting. Survival was demonstrated by lactate clearance, bile production and synthesis of coagulation factors. Conclusions: Liver splitting during normothermic machine perfusion has the potential to revolutionise split liver transplantation. We describe a novel technique that reliably achieves two grafts from a single donor liver. This raises the possibility of semi-elective transplantation, and sophisticated graft assess-ment prior to implant.

Abstract Current perfusion technology only allows livers to be preserved ex-vivo for short periods. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed develop a long-term model of ex-vivo perfusion including a surgical split and simultaneous perfusion of both partial grafts. Our long-term perfusion system included long-term oxygenators, a gas-mixer and a dialysis filter. Human livers declined for transplantation were perfused using a red-cell based perfusate under normothermic conditions (36°C) and then split and simultaneously perfused on separate machines. Ten human livers were split resulting in 20 partial grafts. The median ex-vivo survival was 165 hours (7 days). Long-term graft survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. The grafts that survived > 7 days demonstrated significantly higher bile production, Factor-V production, and hepatic arterial flow and significantly lower microvesicular steatosis. We report reliable long-term ex-vivo perfusion of human livers and demonstrate the ability to split and perfuse these organs using a reproducible protocol. This provides the opportunity for improved assessment of organs and could act as a model for the testing of therapeutics with a matched control.

BACKGROUND:Ex situ machine perfusion facilitates the assessment of livers prior to transplantation. However, currently available markers of liver function poorly predict long-term graft function. Indocyanine green (ICG) is a liver-specific dye which, although common in vivo, has never been comprehensively evaluated for the assessment of graft quality during ex situ machine perfusion. This study aimed to assess the utility of ICG in the ex situ setting.METHODS:Using a customized long-term perfusion system, human livers that were not suitable for transplantation were perfused using a red cell-based perfusate. ICG was delivered into the perfusate on days 0, 1, and 4 to assess ICG clearance (spectrophotometric absorbance at 805 nm) and ICG fluorescence (near-infrared camera).RESULTS:Sixteen partial livers were perfused for a median duration of 172 h (7.2 days). On day 0, the median ICG perfusate disappearance rate (PDR) was 7.5%/min and the median ICG retention at 15 min was 9.9%. Grafts that survived ≥7 days had a significantly higher median ICG PDR on day 0 (14.5%/min vs. 6.5%/min, p = 0.005) but not on days 1 or 4. ICG perfusion demonstrated that long-surviving grafts had a significantly lower median red-value (89.8 vs. 118.6, p = 0.011) and a significantly lower median blue-value (12.9 vs. 22.6, p = 0.045) than short-surviving grafts.CONCLUSION:ICG is a novel marker for the assessment of liver function during ex situ normothermic machine perfusion. ICG PDR and quantitative ICG perfusion can distinguish between long- and short-surviving grafts and demonstrate the utility of ICG in the assessment of graft quality prior to transplant.

Indocyanine green (ICG) is a fluorescent dye taken up and almost exclusively cleared by the liver. Measurement of its clearance and visualization of its fluorescence make it suitable for a number of potential applications in liver transplantation including assessment of liver function and real-time assessment of arterial, venous, and biliary structures. ICG clearance can be used to assess donor graft quality before procurement and graft metabolic function before transplant using normothermic ex vivo machine perfusion. ICG clearance in the post–liver transplantation period is able to predict recipient outcomes with correlations to early allograft dysfunction and postoperative complications. After absorbing light in the near-infrared spectrum, ICG also emits fluorescence at 835 nm. This allows the assessment of vascular patency after reconstruction and patterns of liver perfusion in real time. ICG perfusion patterns after revascularization are also associated with posttransplant graft function and survival. ICG fluorescence cholangiography is routine in a number of centers and acts as an aid to identifying the optimal point of bile duct division during living donor liver transplantation to optimize safety for both donor and recipient. In summary, ICG is a versatile tool and has a number of useful applications in the liver transplantation journey including assessment of liver function, perfusion assessment, and cholangiography. Further research and clinical trials are required to validate and standardize its routine use in liver transplantation.

Introduction: Ischaemia is inevitable during organ preservation and contributes to graft dysfunction after liver transplantation. Early oxygenation of grafts during organ preservation, such as oxygenated washout (OW), has been shown to reduce ischaemic injury. However, the optimal oxygen concentration for OW is unknown. Hyper-oxygenation can induce reactive oxygen species and graft injury. This study compared non-oxygenated preservation with moderate and high oxygen concentrations during OW in rat livers. Methods: Donation after circulatory death livers were procured from 10 rats. Grafts were randomised to three groups: control (non-oxygenated University of Wisconsin Solution (UW)), moderate concentration OW (Mod-OW) (25-28mg dissolved oxygen/L) and high concentration OW(High-OW) (>50mg dissolved oxygen/L). Livers were flushed and stored at 4℃. After 24 hours of cold storage, livers were placed on isolated liver-reperfusion (ILRP) for 120 minutes. Dissolved oxygen in preservation fluid was measured throughout cold storage. Tissue ATP, as a marker of graft viability, was measured at 0, 6 and 24H cold storage, and after ILRP. Liver biochemistry and graft oxygen consumption were measured during ILRP. Results: The control group had lower dissolved oxygen in the preservation fluid than OW groups at 0H and 6H (all p<0.05) (Figure 1A-B). Tissue ATP was similar between groups at 0H. By 6H, grafts in the Mod-OW group had greater tissue ATP than the High-OW group (15.7 vs 8.4 ug/mg, p = 0.01) (Figure 1C). After 24H cold storage, preservation fluid oxygenation and tissue ATP were similar between all groups. During 120 minutes ILRP, the mean oxygen consumption of the Mod-OW group was greater than the High-OW group (47.2 vs 24.4 uL/min/g-liver, p = 0.048) (Figure 1D). No difference in liver biochemistry was observed (Figure 1E-F). Conclusion: Rat livers receiving Mod-OW had increased tissue viability at 6h and oxygen consumption on ILRP compared to High-OW. Our findings suggest that high oxygen concentrations may lead to reduced graft viability. Although OW may be beneficial for organ preservation, oxygen concentrations should be closely monitored to prevent hyper-oxygenation.

Introduction Split liver transplantation (SLT) enables two recipients to be transplanted using a single donor liver; typically, an adult and a child. Despite equivalent long-term outcomes to whole grafts in selected adults, the use of these grafts in high-risk adult recipients with high model for end-stage liver disease (MELD) scores (>= 30), a poor pre-transplant clinical status (ICU or hospital-bound), acute liver failure or retransplantation remains controversial. Methods We retrospectively analyzed all deceased donor adult liver transplants performed between July 2002 and November 2019 at a single high-volume center and performed a propensity score-matched analysis. A subgroup analysis was performed to assess utility of these grafts for high-risk recipients. Results A total of 1090 adult liver transplants were performed, including 155 SLT (14%). Graft survival at 1-, 3- and 5-years were comparable between recipients of split and whole liver grafts (82%, 79% and 74% vs. 86%, 81% and 77%, respectively, log rank P = .537), as was patient survival at 1-, 3- and 5-years. Recipients of split grafts were more likely to have biliary complications and hepatic artery thrombosis, but equivalent long-term survival. Recipients with high MELD scores or a poor pre-transplant clinical status had similar patient and graft survival and complication profiles irrespective of whether they received split or whole grafts. Conclusions SLT is an important method for addressing donor shortages and provides comparable long-term outcomes in adult recipients despite an increase in short-term complications. SLT use in high-risk recipients should be considered to allow for sickest-first allocation policies.

Introduction: Current ex-vivo technology allows the perfusion of an organ only for a number of hours. There is a need for perfusion in the range of days-to-weeks to facilitate sophisticated assessment, recovery and modification of these organs. Normothermic perfusion of livers longer than 1 week has never been previously described. In this study, we aimed to develop a model which reliably maintained the physiological function of human livers for more than 1 week and understand the requirements of these organs for long-term survival. Method: We developed a protocol for long-term organ perfusion using a modified commercial system which included long-term oxygenators, a gas-mixer and a dialysis filter. Human livers not suitable for transplantation were perfused using a human red-cell based perfusate under normothermic conditions (36°C) and then surgically split without interruption to perfusion. The resulting left lateral segment grafts and extended right grafts were then perfused on separate machines for the purpose of long-term survival and individual graft assessment with biochemical and histological markers of liver function (Figure 1). Novel methods for understanding the cause of graft failure of these human livers in the long-term were used including indocyanine green perfusion, microbial cultures, lipidomics, metabolomics and assessment of pro-inflammatory cytokines. Results: Ten livers underwent a conventional split during normothermic perfusion resulting in 20 partial grafts. The median ex-vivo survival was 165 hours, and the longest survival was 328 hours (13 days) (Figure 2). Graft survival was demonstrated by lactate clearance, bile production and production of Factor-V in the long-term. A total of 9/20 (45%) grafts survived for ≥7 days. These grafts demonstrated a significantly higher bile production adjusted to graft weight and a significantly higher level of perfusate Factor-V in the first 48 hours of perfusion (Figure 2). All grafts eventually failed with a predictable and repeatable pattern which include: increasing vascular resistance, unresponsive hypoglycaemia, acidosis and growth of mixed flora microbial contaminants. Conclusion: We report a reliable and repeatable protocol for the long-term ex-vivo perfusion of human livers for more than 1 week. Possible reasons for organ failure during long-term perfusion include overwhelming infection and a failure to meet all long-term metabolic requirements. Future work should aim to overcome these barriers with the goal of preservation for weeks-to-months, if not indefinitely.

We read with interest the article by Gilbo et al1 published in the May issue of Transplantation and the reply by Patrono et al.2 Briefly, the authors report the impact of donor hepatectomy and warm ischemic times on posttransplant outcomes. The study found that graft implantation time, including the interval between portal vein and hepatic artery reperfusion, was not associated with nonanastomotic stricture (NAS) formation. The authors hypothesized that cholangiocyte injury predominantly occurs during organ procurement rather than during implantation. We have previously conducted a study examining bile duct injury in the transplant setting. We examined bile duct biopsies collected before graft reperfusion and after the hepatic artery reperfusion of 45 brain-dead donor grafts from March 2016 to June 2017. Histological injury to the bile duct was assessed as previously described by op den Dries et al.3 We found evidence of peribiliary vascular plexus injury in almost all the prereperfusion biopsy, confirming that cholangiocyte injury occurs during graft retrieval and preservation. However, 22% (n = 10) of postreperfusion biopsies showed worsening severity of the histological bile duct injury. Donor factors and cold ischemic time were not significantly different in grafts that demonstrated worsening histological evidence of bile duct injury after reperfusion. An interval between portal vein and hepatic artery reperfusion >40 min was the only variable associated with worsening histological evidence of bile duct injury after reperfusion (45% versus 15%, P = 0.048) (Figure 1).FIGURE 1.: Interval between portal vein and hepatic artery reperfusion >40 min was associated with worsening peribiliary vascular plexus injury in the postreperfusion biopsy compared with prereperfusion biopsies.It is generally believed that a prolonged suturing time before reperfusion will cause progressive rewarming and subsequently an increased injury of the liver graft. However, Gilbo et al1 showed that the concept of warm ischemic time in the recipient being a major problem is theoretical and perhaps overstated when the difference is measured in minutes. While the warm ischemic time may not correlate with graft function, the sequence of reperfusion may potentially have an impact on cholangiocyte and hepatocyte injury. The ideal technique to reperfuse the graft in liver transplantation remains to be defined. Currently, a surgeon’s choice of a reperfusion technique is based mainly on institutional and personal experience more than scientific evidence. To minimize warm ischemia, most centers use sequential reperfusion, where liver is initially reperfused through portal vein followed by arterial reperfusion. The second most commonly used technique is simultaneous (SIM) reperfusion, where both the portal vein and arterial anastomosis are constructed and reperfused simultaneously. In a previous clinical study, we have demonstrated that microvascular dysfunction and biochemical markers of liver function are influenced by the interval between portal and arterial reperfusion.4 Furthermore, 4 studies compared SIM with sequential reperfusion, and 3 suggested that SIM reperfusion is associated with reduced biliary strictures, but this was not statistically significant in a meta-analysis.5 While warm ischemia time may not significantly impact liver function or NAS, the importance of sequence of reperfusion in determining NAS needs to be defined in larger randomized studies.

In this case report, we preserved human livers for up to 13 days under normothermic conditions using a modified commercial perfusion system. Two whole livers were split into two left lateral segment grafts and two extended right grafts without interruption to blood flow and then perfused on separate machines. Not only does this provide the basis for a meaningful study of liver function in the long term, but this could also facilitate the development of a model of ex situ liver regeneration.