Preservation of non-heart-beating donor livers in extracorporeal liver perfusion and histidine-trytophan-ketoglutarate solution

AIM： To compare the preservation of non-heart- beating donor （NHBD） livers in cold histidine-trytophan- ketoglutarate （HTK） solution and extracorporeal liver perfusion （ECLP）. METHODS： Livers harvested from health pigs were stored for 10 h in cold HTK solution （group A, n = 4） or perfused with oxygenated autologous blood at body temperature （group B, n = 4）. Both groups were then tested on the circuit for 4 h. Bile production, hemodynamic parameters, hepatocyte markers and reperfusion injury of extracorporeal livers were tested in each group. Liver tissues from each group were examined at the end of reperfusion. RESULTS： At 1, 2, 3 and 4 h after reperfusion, bile production, hemodynamic parameters, hepatocyte markers and reperfusion injury of livers in group A were statistically different from those in group B （P 〈 0.05 or P 〈 0.01）. CONCLUSION： ECLP is better than HTK solution to preserve NHBD livers. ECLP can assess the graft viabilitybefore liver transplantation.     

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.     

Cryopreserved mouse hepatocytes retain regenerative capacity in vivo

BACKGROUND & AIMS: Substitution of hepatocyte transplantation for whole liver transplants in selected individuals with liver disease could significantly expand the number of patients to benefit from use of scarce donor livers. However, successful hepatocyte transplantation may require that donor cells retain normal functional and proliferative capabilities and that they be readily available. Banking of cryopreserved hepatocytes would fulfill the latter requirement. Cryopreservation protocols have been developed that minimize hepatocyte injury and allow preservation of metabolic activity. The aim of this study was to assess cryopreserved hepatocyte proliferative capacity in vivo after thawing. METHODS: Fresh and frozen/thawed mouse hepatocytes were transferred separately into the livers of recipient mice with transgene-induced liver disease, an environment that is permissive for clonal expansion of donor cell populations. Fresh and cryopreserved donor cells were compared for their ability to proliferate and replace damaged parenchyma. RESULTS: Although cryopreservation decreased hepatocyte viability, individual viable frozen/thawed hepatocytes demonstrated clonal replicative potential identical to that of fresh hepatocytes. Even after storage for 32 months in liquid nitrogen, transplanted hepatocytes constituting 0.1% of total adult hepatocyte number could repopulate a mean of 32% of recipient liver parenchyma. CONCLUSIONS: These findings suggest that cryopreserved hepatocytes represent an appropriate source of cells for therapeutic hepatocyte transplantation.     

静脉充氧结合抗氧化剂“复苏”无心跳供肝的实验研究

目的：探讨利用静脉充氧“复苏”较长时间无心跳（热缺血30－60min)供体器官技术进行动物原位肝移植的可行性。方法：36头家猪随机分为供体和受体各3组，供体3组为A组（肝脏热缺血15min),B组（肝脏热缺血60min)和C组（肝脏热缺血60min＋冷保存期供氧），进行动物原位肝移植，观察移植动物的5d生存率，肝细胞损害，肝脏合成功能和全身炎症反应指标的变化。结果：B组供肝组在移植后3h内动物全部死亡，A，C组动物存活至预定观察时间；A，C组动物的肝细胞损害，肝脏合成功能和全身炎症反应指标的变化趋势类似，两组间差异无显著性。结论：冷保存期静脉系统供氧对较长时间热缺血损伤的供体肝脏有一定的保护和复苏作用，有可能成先心跳供肝进行肝移植的辅助方法。     

Hepatocyte transplantation program: Lessons learned and future strategies

This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation(HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that:(1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver;(2) Organs with steatosis(≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained;(3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT;(4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and(5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a veryuseful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.     

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats

Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Conclusion: Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications.     

Hepatocyte Isolation From Unused/Rejected Livers for Transplantation: Initial Step Toward Hepatocyte Transplantation,the First Experience From Iran

Background

Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes can be isolated from unused/rejected livers under sterile conditions.

Objectives

The quality of the hepatocytes is very important and the main and initial step in hepatocyte transplantation is hepatocyte isolation. In this study we tried to set up the methods of hepatocyte isolation in order to use the high quality cells in acute liver failure or congenital metabolic disorders.

Materials and Methods

In this study, during a year, hepatocytes were isolated from 7 unused/rejected livers among more than 300 harvested livers in Shiraz University of Medical Sciences. The two step collagenase perfusion method was used under GMP (Good manufacturing practice) for hepatocyte isolation.

Results

Highly quality hepatocytes with high viability and low contamination were isolated. The mean viability was 71.8% ± 21.7. In the first 4 cases microbial contamination by Staphylococci, Diphtheroid and Klebsiella was detected, however the last 3 cases were free of any micro organisms. After 5 weeks of cryopreservation in -140°C, the cell viability was still acceptable.

Conclusions

Hepatocyte isolation can be performed as the main and initial step for cell transplantation from unused/rejected liver. It is the first experience in Iran.     

The capability of isolated hepatocytes and liver slices of donor livers to predict graft function after liver transplantation

AIMS/BACKGROUND: In liver transplantation, adequate function tests for donor livers and transplanted livers are of utmost importance to provide an objective basis for decision-making. Isolated hepatocyte and/or slice preparations from human donor liver tissue may be suitable to test the quality of the organ to be transplanted. METHODS: Surgical waste material remaining after reduced size or split liver transplantation in children was used to prepare slices and isolated hepatocytes. The viability of these preparations as well as drug transport and metabolism functions were determined and related to graft function in 32 liver recipients. RESULTS: The in vitro tests used in the present study apparently did not select non-viable livers. In vitro preparations of the primary non-function grafts which occurred in the investigated group showed normal viability, metabolic and uptake function. CONCLUSION: These results indicate that either the presently used viability tests are not sensitive enough to detect potential organ failure or that other factors besides the hepatocyte viability at the time of transplantation are of paramount importance to the graft function of the recipient, such as complications during and after transplantation or the viability of the non-parenchymal cells.     

Effects of plasma from patients with fulminant hepatic failure on function of primary rat hepatocytes in three-dimensional culture.

BACKGROUND/AIM: As biotechnology continues to advance, a bioartificial liver is expected to be developed for the treatment of patients with fulminant hepatic failure (FHF) whose liver dysfunction is potentially reversible or for providing liver support as a bridge to liver transplantation. While monolayer-cultured hepatocytes rapidly lose their capacity to express many liver-specific functions over time when cultured, spherical-shaped hepatocytes in three-dimensional culture with the use of extracellular matrix components sustain long-term survival by maintaining differentiated hepatocyte functions. The aim of this study was to investigate whether sufficient functions of viable spherical-shaped hepatocytes could be maintained in plasma of patients with FHF in order to use these cells in an extracorporeal system. METHODS: Hepatocyte functions were evaluated under monolayer or three-dimensional culture in FHF plasma. RESULTS: Primary rat hepatocytes on poly-N-p-vinylbenzyl-D-lactonamide (PVLA) formed spheroids even in FHF plasma and maintained their spherical shapes in FHF plasma as long as in medium. Spherical-shaped hepatocytes on PVLA cultured in FHF plasma showed higher activity in albumin secretion, urea formation, and gluconeogenesis than those in normal human plasma or medium. As being cultured in medium, hepatocytes on PVLA cultured in plasma were also superior to cells on collagen in regard to albumin secretion, amino acid metabolism, and gluconeogenesis. CONCLUSIONS: These findings demonstrated that FHF plasma is not toxic to rat hepatocyte spheroids and that hepatocyte spheroids have potential use in the development of a bioartificial liver.     

Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.     

Rapid clearance of transplanted hepatocytes from pulmonary capillaries in rats indicates a wide safety margin of liver repopulation and the potential of using surrogate albumin particles for safety analysis

BACKGROUND/AIMS: Applications of liver repopulation by hepatocyte transplantation require analysis of cell biodistributions, particularly when portasystemic shunting coexists. The aims of this study were to determine the fate of hepatocytes transplanted into the pulmonary vascular bed and to examine whether cell biodistributions could be approximated by convenient surrogates. METHODS: Rat hepatocytes and macroaggregated serum albumin particles of similar sizes were injected into the portal and pulmonary vascular beds of rats, followed by biodistribution, survival and function analyses. RESULTS: Although functionally intact, virtually all hepatocytes were cleared from the pulmonary capillaries within 24 h. Serum albumin levels increased minimally in Nagase analbuminemic rats with or without portacaval shunting to enhance delivery of portal factors to transplanted cells in lungs. Despite intravenous injection of hepatocytes approaching >1x10(9) cells in humans, the hemodynamic changes were limited to transient increases in right atrial pressures. The hepatocyte distributions in specific vascular beds were largely reproduced by macroaggregated human serum albumin particles. CONCLUSIONS: Incidental intrapulmonary cell translocations during liver repopulation will have a wide safety margin. Use of macroaggregated serum albumin particles as surrogates for initial short-term biodistribution and safety analysis will advance hepatocyte transplantation, as the cost of GLP-certified laboratories and consumption of scarce donor livers will be avoided.     

Intermittent ischaemia maintains function after ischaemia reperfusion in steatotic livers

Background:

Ischaemic preconditioning (IPC) and intermittent ischaemia (INT) reduce liver injury after ischaemia reperfusion (IR). Steatotic livers are at a higher risk of IR injury, but the protection offered by IPC and INT is not well understood. The aim of the present study was to determine the effectiveness of IPC and INT in maintaining liver function in steatotic livers.

Material and methods:

A model of segmental hepatic ischaemia (45 min) and reperfusion (60 min) was employed using lean and obese Zucker rats. Bile flow recovery was measured to assess dynamic liver function, hepatocyte fat content quantified and blood electrolytes, metabolites and bile calcium measured to assess liver and whole body physiology. Liver marker enzymes and light and electron microscopy were employed to assess hepatocyte injury.

Results:

IPC was not effective in promoting bile flow recovery after IR in either lean or steatotic livers, whereas INT promoted good bile flow recovery in steatotic as well as lean livers. However, the bile flow recovery in steatotic livers was less than that in lean livers. In steatotic livers, ischaemia led to a rapid and substantial decrease in fat content. Steatotic livers were more susceptible to IR injury than lean livers, as indicated by increased blood ALT concentrations and major histological injury.

Conclusion:

INT is more effective than IPC in restoring liver function in the acute phase of IR in steatotic livers. In obese patients, INT may be useful in promoting better liver function after IR after liver resection.     

The hepatic microcirculation in the isolated perfused human liver

In cirrhosis, capillarization of sinusoids could result in impaired exchanges between the hepatocytes and the blood perfusing the liver and contribute to liver failure irrespective of the metabolic capacity of the liver. To characterize anomalies of the hepatic microcirculation, we used the multiple-indicator dilution approach in isolated perfused livers obtained from patients with cirrhosis at the time of transplantation, and from organ donors with normal or near-normal livers or hepatic steatosis. In organ donors, the sinusoidal volume and the permeability of sinusoids to albumin, sucrose, and water were found to be comparable to that of normal dog and rat livers. The sinusoidal volume and the extravascular volume (EVV) accessible to diffusible tracers were larger after hepatic artery than after portal vein injection, probably because of an unshared arterial sinusoidal bed. In cirrhotic livers, two kinds of alterations were found: the appearance of a barrier between the sinusoids and the hepatocytes (capillarization) and intrahepatic shunts. The extravascular space accessible to albumin decreased with increasing severity of cirrhosis, and the diffusion of sucrose in the space of Disse showed a barrier-limited pattern, instead of the normal flow-limited behavior. In cirrhotic livers, a correlation was found between the hepatic extraction of indocyanine green (ICG) and the extravascular space accessible to albumin (r = .84, P < .05), suggesting that the impaired access of this protein-bound dye to the hepatocyte surface contributed to its impaired elimination. Intrahepatic shunts were found between portal and hepatic vein (21% +/- 16% of portal flow), but not between hepatic artery and hepatic veins. We conclude that (1) the behavior of diffusible tracers in human livers with normal liver architecture is comparable to that reported in normal animals; (2) the permeability of sinusoids in cirrhotic livers is abnormal, (3) permeability changes are related to changes in liver function in cirrhosis. (Hepatology 1996 Jan;23(1):24-31)     

成人脂肪肝整肝肝细胞分离及大量冻存的实验研究

目的建立成人脂肪肝整肝肝细胞的分离以及人肝细胞大量冻存技术,为生物人工肝提供稳定的人肝细胞来源。方法采用胶原酶经肝静脉逆行灌注的方法分离成人重度脂肪肝的整肝肝细胞,并比较常规冻存和程序冻存后肝细胞在细胞活性、贴壁率、LDH漏出量及白蛋白合成能力的差异。结果采用添加N-乙酰半胱氨酸(NAC)的胶原酶灌注液分离肝细胞的产量为(7.4±0.5)×10~6 cells/g肝组织,活性为(81.4±3.4)%,而未添加NAC组的肝细胞产量为(5.6±0.8)×10~6 cells/g肝组织和活性为(67.3±5.0)%,差异均有统计学意义(P0.05)。分离的人肝细胞采用程序冻存后在细胞活性、贴壁率及白蛋白合成能力方面均相应高于常规冻存组(P0.05),LDH漏出量低于常规冻存组(P0.05)。结论应用添加NAC的胶原酶灌注液经肝静脉逆行灌注可提高脂肪肝整肝肝细胞分离的活性及产量,采用程序冻存的方法可以提高冻存人肝细胞的活性,满足生物人工肝对肝细胞的需要。     

Hepatocyte transplantation in rats with decompensated cirrhosis

Hepatocyte transplantation improves the survival of laboratory animals with experimentally induced acute liver failure and the physiological abnormalities associated with liver-based metabolic deficiencies. The role of hepatocyte transplantation in treating decompensated liver cirrhosis, however, has not been studied in depth. To address this issue, cirrhosis was induced using phenobarbital and carbon tetrachloride (CCL(4)) and animals were studied only when evidence of liver failure did not improve when CCL(4) was held for 4 weeks. Animals received intrasplenic transplantation of syngeneic rat hepatocytes (G1); intraperitoneal transplantation of syngeneic rat hepatocytes (G2); intraperitoneal transplantation of a cellular homogenate of syngeneic rat hepatocytes (G3); intraperitoneal transplantation of syngeneic rat bone marrow cells (G4); or intrasplenic injection of Dulbecco's modified Eagle medium (DMEM) (G5). After transplantation, body weight and serum albumin levels deteriorated over time in all control (G2-G5) animals but did not deteriorate in animals receiving intrasplenic hepatocyte transplantation (G1) (P <.01). Prothrombin time (PT), total bilirubin, serum ammonia, and hepatic encephalopathy score were also significantly improved toward normal in animals receiving intrasplenic hepatocyte transplantation (P <. 01). More importantly, survival was prolonged after a single infusion of hepatocytes and a second infusion prolonged survival from 15 to 128 days (P <.01). Thus, hepatocyte transplantation can improve liver function and prolong the survival of rats with irreversible, decompensated cirrhosis and may be useful in the treatment of cirrhosis in humans.     

N-acetylcysteine and liberase improve success of hepatocyte isolation and viability of hepatocytes isolated from normal and diseased liver

BackgroundSuccessful hepatocyte isolation is crucial for development of cellular transplantation and biochemical research. Most researchers isolate hepatocytes from surplus donor tissue or normal tissue removed during resection of liver tumours. However, most tissue available for research is from explanted diseased liver and donor tissue rejected for transplant. We previously described our experience of hepatocyte isolation using liberase from such livers with a success rate of 51% and median viability of 40%. Liberase is a highly purified collagenase that has been shown to improve the viability of isolated porcine hepatocytes. N-acetylcysteine (NAC) has been shown to improve the viability of human hepatocytes isolated from steatotic donor tissue. The aim of this study was to determine the effect of both reagents in combination on the outcome of hepatocyte isolation from normal and diseased liver.MethodsHepatocytes were isolated from 30 consecutive liver specimens as previously described (old protocol). A further 30 consecutive liver specimens were perfused with buffer containing NAC and standard collagenase substituted by liberase (new protocol). Success was defined as maintenance of cell adhesion and morphology for 48 h and/or their successful use in laboratory studies. Mann-Whitney tests were used to compare results. Fisher's exact test was used for categorical data.FindingsBaseline factors were similar for both groups. The delay to tissue processing was slightly less in the new protocol group (median 2 h vs 4 h, p=0·007). The success rate improved from 40% (12/30) with the old protocol to 70% (21/30) with the new protocol (p=0·037), and the median viable cell yield increased from 7·3 × 10⁴ to 28·3 × 10⁴ cells per g tissue (p=0·003). After multivariable analysis adjusting for the difference in time delay, the success rate (p=0·014) and viable cell yield per g tissue (p=0·001) remained significantly improved.InterpretationNAC and liberase greatly improve the success of hepatocyte isolation and result in a significantly higher viable cell yield. Use of these agents may improve the availability of hepatocytes for transplantation as well as laboratory research.FundingUK Medical Research Council.     

Hepatocyte and Kupffer cell function after liver transplantation in the rat –in vivo evaluation with dynamic scintigraphy

Abstract: In vivo physiological measurements of hepatocyte and Kupffer cell function after liver transplantation are desirable. Orthotopic liver transplantation was performed in 54 rats. Hepatocyte and Kupffer cell function were measured with dynamic liver scintigraphy. Hepatic clearance of ^99mTc-Nanocoll (%/min), an albumin colloid phagocytosed by the Kupffer cells, was used to evaluate Kupffer cell function. Hepatic clearance of ^99mTc-IODIDA (%/min), an imino-diacetic-acid taken up and secreted by the hepatocytes, was used to evaluate the hepatocyte function. Hepatic clearance in control rats was 27±2 %/min for Nanocoll and 30±3 %/min for IODIDA. After syngenic liver transplantation, without rejection, there was a rise in Nanocoll clearance (34±2 %/min p<0.01) after 3 weeks, but no change in IODIDA clearance (32±3 %/min N.S.). After syngenic liver transplantation with preservation time prolonged to 16 h, there were no changes in IODIDA or Nanocoll clearance 1 day after transplantation. Both IODIDA (11±2 %/min) and Nanocoll clearance (22±2 %/min) were decreased (p<0.001) during rejection after allogenic transplantation. An in vivo method of measuring the hepatocyte and Kupffer cell function in the transplanted liver is described. Kupffer cell function was increased after syngenic liver transplantation. Kupffer cell and hepatocyte function were decreased during rejection. Dynamic liver scintigraphy seems a suitable procedure for examining liver injury after liver transplantation in the experimental setting.     

Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation

BACKGROUND & AIMS: Hepatocyte transplantation has been proposed as an alternative to liver transplantation for the treatment of hepatic failure. A major limitation to this form of therapy is the availability of human livers as a source of hepatocytes. The use of porcine hepatocytes might address this problem; however, xenogeneic hepatocytes are thought to be functionally incompatible across species and susceptible to irreversible rejection. METHODS: Liver cirrhosis was induced with phenobarbital and carbon tetrachloride. Only rats with decompensated liver failure that did not correct 4 weeks after the discontinuation of carbon tetrachloride were subjected to intrasplenic rat or porcine hepatocyte transplantation. The immunologic integrity of cirrhotic rats was assessed by allogeneic skin grafting, and the immune response to transplanted porcine hepatocytes was assessed by enzyme-linked immunosorbent assay. RESULTS: Porcine hepatocytes restored metabolic function and prolonged the survival of cirrhotic rats, as well as rat hepatocytes. Cirrhotic rats retained the ability to reject allogeneic skin grafts and showed an immune response to the engrafted hepatocytes. Despite this, survival of transplanted porcine hepatocytes was accepted in cirrhotic rats for a period of weeks without immunosuppression. Conventional immunosuppression with FK506 allowed successful retransplantation with hepatocytes from a second porcine donor. CONCLUSIONS: Hepatocytes transplanted between widely divergent species can function to correct liver failure in cirrhotic rats and prolong their survival. Conventional immunosuppression allows long-term functioning of xenogeneic hepatocyte retransplants and suggests that hepatocyte xenotransplantation might be useful as a bridge to liver transplantation and could potentially provide long-term hepatic support.     

Ischemic cholangiopathy after liver transplantation

Orthotopic liver transplantation (OLT) has evolved over the last forty years from an experimental endeavor to standard of care therapy for many patients with end stage hepatic disease. Many technical advances have contributed to the current success of OLT, but surgical complications, especially involving the biliary reconstruction, remain a morbid problem. Biliary complications after OLT include leaks and strictures. Strictures may be anastomotic or intrahepatic and diffuse, as seen in cases of hepatic artery thrombosis. Current efforts to expand the limited donor pool include the use of non-heart beating donors. The organ procurement process in these donors entails an increased period of warm ischemia and results with non-heart beating donor grafts have been mixed. It is now appreciated that there is an increased incidence of subsequent diffuse biliary stricturing or "ischemic cholangiopathy" in recipients of these organs. Animal models of this phenomenon and potential therapeutic strategies targeted at ischemic cholangiopathy are being developed with potential applicability to non-heart beating donation and will be the focus of this review.