Transplantation Of Isolated Hepatocytes Research Articles

Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7) hepatocytes or phosphate buffered saline through the portal vein or into the spleen and were sacrificed after 48 hours. Alanine aminotransferase levels measured within the experiment did not differ between groups at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. These data demonstrate the feasibility and efficacy of hepatocyte transplantation in the liver or spleen in a mild acetaminophen-induced hepatotoxicity model.

An Update on Hepatic Stem Cells: Bench to Bedside

Liver failure results in impairment of many functions and dependent organs such as brain and kidneys begin to fail, reducing the chance of recovery even further. Orthotopic liver transplantation (OLTx) is the only treatment that improves the survival rate in patients with liver failure. Liver Transplantation (LT), including orthologous liver transplantation (OLT), cadaveric LT, split LT, living donor LT (LDLT) brings hopes to patients with these diseases. Globally, 1.4 million deaths occur annually as a result of chronic liver diseases. The reasons for this high death toll include unavailability of healthy liver donor and highly expensive liver transplantation treatment. Furthermore, some other factors such as operative risks and post-transplant rejection are major limitation of OLT. Isolated hepatocyte transplantation is emerging as alternative bridge support till the healthy donor is arranged. Mature hepatocytes have several drawbacks such as low proliferation both in vitro and in vivo, low viability after cryopreservation, and requirement of large number of cells for infusion. The studies on isolation of hepatic progenitors have shown promising results to overcome these limitations. These cells possess higher proliferative capacity, are less immunogenic and more resistant to cryopreservation, and ischemic injury; properties that could enhance their engraftment within the recipient liver. The hepatic progenitors have been isolated from the intra-hepatic sources and extra-hepatic sources. Fetal cells are one of the ideal sources of hepatic stem/progenitor cells. Autologous bone marrow stem cell transplantation in patients with cirrhosis has shown promising result.

Hepatocyte transplantation.

Isolated hepatocyte transplantation has long been recognized as a potential treatment for life-threatening liver disease. The basis for proceeding with clinical trials has been established by the extensive laboratory work in animal models. Human hepatocyte transplantation has been applied in individual cases and very small, uncontrolled series. Data, although sparse, demonstrate the safety and feasibility of this approach and are supportive, if less than conclusive, of effectiveness. The experience of hepatocyte transplantation in the laboratory and clinical arenas is reviewed and discussion will examine what is believed to be the primary cause for the slow growth of this technology in the clinical setting, namely a severe shortage of usable primary human hepatocytes. The potential of isolated hepatocyte transplantation remains largely untapped and awaits alternate sources of cells for transplantation other than those from discarded human cadaveric livers.

Review article: a medicine based on cell transplantation -- is there a future for treating liver diseases?

Cell-based therapies, comprising isolated hepatocyte transplantation, ex vivo gene therapy and bioartificial liver devices, may actually design a new scenario in the treatment of patients with liver failure. Recent advances in the liver repopulation field and the considerable excitement surrounding the use of haematopoietic stem cells for a 'regenerative medicine', allow us to see under a new light the alternative cell sources actually under investigation. In particular, the major drawbacks and the most important advantages of xenogenic primary cells, tumour-derived cell lines, immortalised hepatocytes and stem cells in the wide range of experimental and clinical applications are presented. Even if up to now none of them represent a 'gold standard' in the clinical practice, the intersecting advances in the development of mechanical components of artificial devices and in the liver biology and bioengineering will open tantalising possibilities to treat patients with liver failure, by tailoring the therapeutic choice on the basis of the aetiology and the stage of liver disease.

Extracorporeal support and hepatocyte transplantation in acute liver failure and cirrhosis.

The relative shortage of donor organs and lack of immediate availability mean that many patients with acute liver failure die before orthotopic liver transplantation can be performed. An effective temporary liver support system could improve the chance of survival with or without a transplant being ultimately carried out. Recent technological advances resulting in improved maintenance of hepatocyte viability and function in culture and bioreactor designs which facilitate adequate perfusion of the cellular component and removal of products of cellular metabolism have led to the development of a number of bioartificial devices for liver support. Three such devices have undergone preliminary clinical evaluation in the setting of acute liver failure, with a statistically significant reduction in raised intracerebral pressure along with improvements in consciousness level and some biochemical parameters associated with treatment with one of these. Several other devices with different characteristics have shown promise in vitro and/or in animal models but await clinical evaluation. Several new totally artificial systems have also been described, along with the emergence of isolated hepatocyte transplantation, with reports of successful 'bridging' to liver transplantation. Controlled trials on a multicentre basis in well-defined patient groups and with standardized outcome measures will be required to properly evaluate the clinical value of each of these approaches to providing liver support in acute liver failure and cirrhosis. A better understanding of mechanisms underlying multiorgan failure and of factors inhibiting liver regeneration, thereby allowing a more targeted approach, will be essential to the further development of effective liver support strategies in these settings.

Pediatric liver disease

Pediatric hepatology has advanced greatly over the past few years. Many more children with severe liver disease are now surviving into adulthood. There is a number of conditions not seen in adult practice that have been the focus of research efforts, and many of these efforts have borne fruit in the past year. Disorders characterized by intrahepatic cholestasis in particular have now been substantially unravelled, and this work has elucidated a great deal about hepatic physiology. Liver-directed gene therapy is on the threshold of human application. This research has been facilitated by excellent animal models and the advent of isolated hepatocyte transplantation. Relatively common conditions such as biliary atresia, however, remain largely unexplained and the viral hepatitides have no effective form of treatment. It remains a hope that our increase in knowledge in the fields of genetics and immunology will translate into advances in therapy.

HEPATOCYTE TRANSPLANTATION IMPROVES LIVER FUNCTION AND PROLONGS SURVIVAL IN RATS WITH DECOMPENSATED LIVER CIRRHOSIS

179 Introduction: Isolated hepatocyte transplantation (HTX) has been shown to improve the survival of laboratory animals with experimentally-induced acute liver failure and to improve the physiologic abnormalities associated with liver-based metabolic deficiencies. The role of HTX in prolonging survival and improving the liver function of animals with decompensated liver cirrhosis, however, has not been adequately studied. In order to address this issue, HTX was performed in rats following induction of stable decompensated liver cirrhosis (LC). Methods: Liver cirrhosis was induced using phenobarbital (PhB) and carbon tetrachloride (CCL4). To avoid the regression of cirrhosis known to follow discontinuation of CCL4, CCL4 was continued until physiologic abnormalities did not improve when CCL4 was held for 4 weeks and the dose was adjusted based on changes in body weight. Twenty five rats developed irreversible ascites and maintained a total bilirubin (TB) >0.5 mg/dl, albumin <2.6 g/dl, prothrombin time (PT) >14.5 sec, and ammonia (NH3) >145 mmol/l. Rats were randomly divided into the following groups: Group 1 (G1: n=5), intrasplenic transplantation of 50×106 primary syngeneic rat hepatocytes; Group 2 (G2: n=5), intraperitoneal transplantation of 200×106 primary syngeneic rat hepatocytes; Group 3 (G3: n=5), intraperitoneal transplantation of a cellular homogenate of 200×106 primary syngeneic rat hepatocytes; Group 4 (G4: n=5), intraperitoneal transplantation of 200×106 syngeneic rat bone marrow cells; and Group 5 (G5: n=5), intrasplenic injection of 1 ml DMEM. Results: Following transplantation, body weight and serum albumin deteriorated over time in all control (G2-G5) animals but did not deteriorate in animals receiving intrasplenic HTX (G1) (p<0.01). Similarly, laboratory tests and hepatic encephalopathy (HE) score remained abnormal in control (G2-G5) animals but significantly improved toward normal in G1 animals. At 35d post-HTX, all measures of liver decompensation were significantly different: PT (G1: 14.0±0.7 sec vs G2-G5: 21.6±1.7 sec), TB (G1: 0.54±0.11 vs G2-G5: 0.99±0.12 mg/dl), NH3 (G1: 122 ± 20 vs G2-G5: 217±17 mmol/L) and HE score (G1: 11.0±2.3 vs G5: 4.8±0.5, no HE=15) (P <0.01). Moreover, survival of animals receiving intrasplenic HTX was significantly prolonged compared to all other groups from 40.0± 4.5 d to 76.6± 4.4 d (G1 vs G2-G5, P <0.01). Postmortem examination revealed severe portal hypertension and histological evidence of micronodular cirrhosis in all rats. Conclusions: Intrasplenic HTX can improve liver function and prolong the survival of rats with irreversible, decompensated liver cirrhosis and may be useful in the treatment of cirrhosis in man.

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts.

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells. (Hepatology 1997 Nov;26(5):1287-95)

Selective intraportal transplantation of DiI-marked isolated rat hepatocytes.

Transplantation of isolated hepatocytes is a promising alternative to orthotopic liver transplantation in experimental animal models with acute hepatic failure and hereditary enzyme defects. Conventional light microscopy identification of hepatocytes within recipient livers has been limited due to the inability to distinguish between donor and recipient liver cells. In this study, we labeled hepatocytes intracellularly with the fluorescent dye DiI-18 prior to selective intraportal or intrasplenic transplantation. Syngeneic LEW rat hepatocytes were isolated and 2 x 10(7) fluorescence-labeled cells were transplanted by intraportal infusion selectively into 2/3 of the recipient liver lobules to avoid lethal portal hypertension. Rats were sacrificed on postop days 1, 3, 5, 10, 20, and 40. Histological examination was performed using light and fluorescence microscopy counterstained by light green dye. The quantity of transplanted hepatocytes residing within the recipient liver was determined by FACS analysis after enzymatic digestion of the recipient liver lobules. Engrafted hepatocytes were identified in the periportal regions of transplanted liver lobules. The stained hepatocytes were retrieved up to 20 days postop using fluorescent microscopy. Using FACS analysis the number of labeled hepatocytes was found to diminish over time following transplantation from 2.1% on postop day 1 to 0.5% on day 10. Labeled hepatocytes transplanted into the spleen were retrieved in clusters up to 20 days postop (the last day of observation). Furthermore, the migration of labeled hepatocytes from spleen to liver parenchyma was observed following intrasplenic transplantation. However, after selective intraportal transplantation, only fluorescent debris was found in splenic and pulmonary tissue upon examination of various organs. This article describes the method of fluorescent labeling of rat hepatocytes and reports the feasibility and limitations of using DiI-18 as a marker.

Treatment of Fulminant Hepatic Failure – Is There Light at the End of the Tunnel?

A lot of research has been done on fulminant hepatic failure (FHF), mainly because of the high mortality associated with it. Many centres continue to search actively for the ideal medical treatment for FHF because liver transplantation is often difficult in these very ill patients. The many forms of therapy that have been tried and reported in case reports, small clinical trials and the occasional randomized controlled trial are reviewed, as are the current data on bioartificial livers and isolated hepatocyte transplantation. The ideal treatment remains elusive, and more work needs to be done to find a highly effective form of therapy for these very ill FHF patients.

Selective intraportal hepatocyte transplantation in analbuminemic and Gunn rats.

Although significant progress has been achieved in isolated hepatocyte transplantation, the optimal site of cell implantation has not yet been determined. We have developed a novel experimental method of intraportal hepatocyte transplantation that allows easy assessment of the morphology and function of transplanted hepatocytes. Donor hepatocytes were harvested from Sprague-Dawley rats by in situ EDTA/collagenase perfusion. Fifteen recipient Nagase analbuminemic rats (NAR) underwent cannulation of the gastroduodenal vein under ether anesthesia. Either the posterior or anterior liver lobes were selectively infused with cells by occluding the portal venous supply of the nontransplanted liver lobes. Normal donor hepatocytes (2 x 10(7)) suspended in normal saline were infused over 1 min (4 ml). Recipients were treated with cyclosporine for the duration of the experiment. Plasma albumin levels were determined by ELISA, before and at various intervals after transplantation. In NAR rats transplanted with normal hepatocytes, there was a significant (P < 0.003) and sustained (12 weeks) increase in plasma albumin levels. Control NAR rats transplanted with NAR hepatocytes (n = 8) showed no significant changes in plasma albumin levels. Similarly, normal Wistar hepatocytes were infused intraportally into the posterior lobes of Gunn rats (n = 4), which lack the ability to conjugate bilirubin. Pre- and posttransplantation bile was collected following bile duct cannulation. Bile analysis by HPLC, demonstrated a significant (P = 0.04) increase in the level of bilirubin conjugates following transplantation and a corresponding decrease in total serum bilirubin (P = 0.04). Our experimental data demonstrate that direct selective intraportal infusion of hepatocytes is an effective technique of hepatocyte transplantation in the rat.

Intrasplenic hepatocyte transplantation: evaluation of DNA synthesis and proliferation in auxiliary transplanted cells.

The value of isolated hepatocyte transplantation as a temporary support in acute hepatic failure remains controversial regarding the functional capacities of freshly isolated and transplanted hepatocytes. To evaluate the survival rate of intrasplenically transplanted liver cells and their response to a proliferation stimulus like partial hepatectomy, 3H-thymidine incorporation into hepatic and auxiliary transplanted hepatocyte DNA was determined. The survival rate of intrasplenically transplanted hepatocytes was evaluated by analyses of m-albumin-RNA within the splenic tissue and compared to the morphological findings. The histological results show a marked decrease (greater than x 100) of intrasplenically transplanted hepatocytes within 1 week after injection. The amount of surviving cells then remained constant for 3 months without any signs of proliferation. After partial hepatectomy a stimulation of hepatic regeneration was observed in the remaining liver tissue but not in auxiliary transplanted hepatocytes. M-albumin-RNA determination of auxiliary transplanted hepatocytes revealed a decrease of m-albumin-RNA concentration of greater than 100 times within 24 h after transplantation indicating early cell necrosis of the transplanted cells. Since intrasplenically transplanted hepatocytes underwent an early cell necrosis without any evidence for a directly postoperatively inducible cell proliferation, it is concluded that a sufficient metabolic support in acute hepatic failure cannot be taken into consideration.

Monitoring of the rejection of intrasplenic isolated hepatocyte transplantation by the technetium 99m-imino diacetic acid (Tc 99m IDA) derivatives study

Monitoring of the rejection of intrasplenic isolated hepatocyte transplantation by the technetium 99m-imino diacetic acid (Tc 99m IDA) derivatives study