Historical aspects of hepatocyte transplantation

Hepatocyte transplantation has been used for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure and as a "cellular therapy" for patients with genetic defects in vital liver functions. The aim of this paper was to discuss the basic research that led to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.     

Engraftment measurement in human liver tissue after liver cell transplantation by short tandem repeats analysis

Hepatocyte transplantation has been proposed as a technique for bridging patients to whole-organ transplantation, for providing metabolic support during liver failure, and for replacing whole-organ transplantation in certain metabolic liver diseases. Assessment of hepatocyte engraftment has been difficult to measure, and the degree of engraftment needed to correct various liver disorders is still unknown. A sensitive, simple, and specific method of monitoring engraftment of transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration using short tandem repeats (STRs) was evaluated. The analytical sensitivity of the test was evaluated using DNA mixing curves and established as 0.5% (percentage of donor DNA/ recipient DNA). Sex-matched and mismatched cases were included during the validation. The clinical evaluation of the assay was performed using liver samples from two patients who underwent hepatocyte transplantation. We concluded from this study that the AmpFLSTR Profiler Plus PCR Amplification Kit, a well-established technique in forensic medicine, is specific, sensitive, and a reproducible assay for measurement of engraftment after hepatocyte transplantation in both sex-matched and sex-mismatched cases.     

Trasplante celular hepático: un nuevo tratamiento en las enfermedades hepáticas

Liver transplantation has been remarkably effective in the treatment in patients with end-stage liver disease. However, disparity between solid-organ supply and increased demand is the greatest limitation, resulting in longer waiting times and increase in mortality of transplant recipients. This situation creates the need to seek alternatives to orthotopic liver transplantation.Hepatocyte transplantation or liver cell transplantation has been proposed as the best method to support patients.The procedure consists of transplanting individual cells to a recipient organ in sufficient quantity to survive and restore the function. The capacity of hepatic regeneration is the biological basis of hepatocyte transplantation. This therapeutic option is an experimental procedure in some patients with inborn errors of metabolism, fulminant hepatic failure and acute and chronic liver failure, as a bridge to orthotopic liver transplantation.In the Hospital La Fe of Valencia, we performed the first hepatocyte trasplantation in Spain creating a new research work on transplant program.     

Bioartificial liver support devices: historical perspectives

Fulminant hepatic failure (FHF) is an important cause of death worldwide. Despite significant improvements in critical care therapy there has been little impact on survival with mortality rates approaching 80%. In many patients the cause of the liver failure is reversible and if short-term hepatic support is provided, the liver may regenerate. Survivors recover full liver function and a normal life expectancy. For many years the only curative treatment for this condition has been liver transplantation, subjecting many patients to replacement of a potentially self-regenerating organ, with the lifetime danger of immunosuppression and its attendant complications, such as malignancy. Because of the shortage of livers available for transplantation, many patients die before a transplant can be performed, or are too ill for operation by the time a liver becomes available. Many patients with hepatic failure do not qualify for liver transplantation because of concomitant infection, metastatic cancer, active alcoholism or concurrent medical problems. The survival of patients excluded from liver transplantation or those with potentially reversible acute hepatitis might be improved with temporary artificial liver support. With a view to this, bioartificial liver support devices have been developed which replace the synthetic, metabolic and detoxification functions of the liver. Some such devices have been evaluated in clinical trials. During the last decade, improvements in bioengineering techniques have been used to refine the membranes and hepatocyte attachment systems used in these devices, in the hope of improving function. The present article reviews the history of liver support systems, the attendant problems encountered, and summarizes the main systems that are currently under evaluation.     

Monitoring of Intrasplenic Hepatocyte Transplantation for Acute-on-Chronic Liver Failure: A Prospective Five-Year Follow-Up Study

Background

Acute-on-chronic liver failure (ACLF) is defined as an acute deterioration of chronic liver disease. Intrasplenic hepatocyte transplantation is increasingly recognized as a treatment for liver failure and genetic metabolic liver diseases. We describe our experience of intrasplenic hepatocyte transplantation in a small cohort of patients as bridge therapy or as an alternative to orthotopic liver transplantation (OLT).

Methods

Seven patients with ACLF with an expected survival of less than 8 weeks were enrolled into the study. The donor hepatocytes were collected from 2 healthy males and cryopreserved. Donor hepatocytes were transplanted into the spleen of recipients via catheterization of the femoral artery. All patients were followed up for 5 years or to death.

Results

A total of (4.2–6.0) × 10¹⁰ hepatocytes were harvested from the 2 donors' livers and their survival after recovery from the frozen stock was 63% ± 2.8% and 73.5% ± 3.2%, respectively. Following intrasplenic hepatocyte transplantation, 3 patients fully recovered from liver failure, 1 survived and subsequently underwent OLT, and the remaining 3 patients died between 2.5 and 12 months after intrasplenic hepatocyte transplantation. At month 48 post–intrasplenic hepatocyte transplantation, living hepatocyte signals were observed in the spleen using magnetic resonance imaging (MRI) with gadobenate dimeglumine (Gd-BOPTA).

Conclusions

Intrasplenic hepatocyte transplantation is a promising therapy for liver failure that may reduce mortality rates among patients with end-stage liver disease awaiting OLT. Conceivably, intrasplenic hepatocyte transplantation may be considered an alternative to OLT for patients with acute liver failure. MRI (Gd-BOPTA) is a useful tool for detecting living hepatocytes in the spleen after intrasplenic hepatocyte transplantation.     

Auxiliary Liver Transplantation and Bioartificial BridgingProcedures in Treatment of Acute Liver Failure

The aim of this review is to discuss the place of auxiliary liver transplantation (ALT) and other cellular based bridging procedures such as hepatocyte transplantation, ex vivo liver perfusion, and bioartificial livers, in the treatment of acute liver failure, vis à vis conventional orthotopic liver transplantation. Hepacocyte transplantation, and ex vivo pig or human liver perfusion are still experimental procedures. Bioartificial livers using human tumoral hepatocytes or porcine hepatocytes have been used in clinical situations as a bridge to transplantation, i.e. to gain the time required to find a high-quality graft for conventional or auxiliary liver transplantation. None of these techniques have yet proved capable of keeping a patient alive long enough for the native liver to recover. Conversely, ALT has been shown to be effective in the treatment of acute liver failure and now appears to be a satisfactory bridging procedure pending native liver (NL) regeneration. We report personal experience of 18 ALT procedures performed in 17 patients between October 1992 and December 1999. The ALT procedure was indicated when patients met criteria for conventional transplantation; it was ultimately selected when a fresh frozen biopsy of the NL did no show any fibrosis. Six patients died within the first 2 postoperative months. The remaining 11 patients are alive, with a follow-up ranging from 2 to 7 years. Regeneration of the NL occurred in 11 of the 17 patients (65%) and in 8 of the 11 survivors (72%), 6 of whom have permanently stopped immunosuppressive therapy. We conclude that liver failure should no longer be handled outside centers where all types of transplantation can be offered, and where innovative therapies such as hepatocyte transplantation and extracorporeal liver-assist devices are being developed and evaluated.     

Hepatic Assist: Present and Future

Fulminant hepatic failure due to acute massive liver cell necrosis is a complex pathophysiological entity, and treatment is still unsatisfactory. Artificial liver supports such as hemodialysis, hemoperfusion, and plasmapheresis have recently been used clinically to treat fulminant hepatic failure. However, survival rate has not improved as expected, although the consciousness of the patient has improved frequently. In this article the present status of clinical artificial liver support and basic research of hybrid artificial liver will be discussed. Moreover, the future aspects of total artificial liver support and hepatocyte transplantation for chronic liver failure will be introduced.     

Extracorporeal liver perfusion using human and pig livers for acute liver failure

BACKGROUND: Patients with fulminant hepatic failure (FHF) often die awaiting liver transplantation. Extracorporeal liver perfusion (ECLP) has been proposed as a method of "bridging" such patients to transplantation. We report the largest experience to date of ECLP using human and porcine livers in patients with acute liver failure. METHODS: Patients with FHF unlikely to survive without liver transplantation were identified. ECLP was performed with human or porcine livers. Patients underwent continuous perfusion until liver transplantation or withdrawal of support. Two perfusion circuits were used: direct perfusion of patient blood through the extracorporeal liver and indirect perfusion with a plasma filter between the patient and the liver. FINDINGS: Fourteen patients were treated with 16 livers in 18 perfusion circuits. Nine patients were successfully "bridged" to transplantation. ECLP stabilized intracranial pressure (ICP) and cerebral perfusion pressure (CPP). Arterial ammonia levels fell from a median of 146 to 83 micromol/liter within 12 hr and this reduction was maintained at least 48 hr. Pig and human ECLP lowered ammonia levels equally. Serum bilirubin levels also fell from a median of 385 to 198 micromol/liter over the first 12 hr but the response was not sustained as well with porcine livers. There was no immunological benefit to using the the filtered perfusion circuit. INTERPRETATION: These data demonstrate that ECLP is safe and can provide metabolic support for comatose patients with fulminant hepatic failure for up to 5 days. While labor and resource intensive, this technology is available to centers caring for patients with acute liver failure and deserves wider evaluation and application.     

Bioengineering of liver assist devices

Over 30 000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver device could provide vital support to a liver failure patient until a donor liver was available or until the patient's own liver regenerated. Although it is unclear which liver-specific functions must be provided by such a device to be effective, a constant challenge has been to obtain stable, well-differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased device efficiency. In this overview, the authors describe the critical issues involved in bioartificial liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat-plate bioartificial liver device with an internal membrane oxygenator. Received: March 20, 2002 / Accepted: April 15, 2002 Acknowledgment. The authors thank Dr. Harihara Baskaran for the preparation of Figure 2. This work was partially funded by grants from the National Institutes of Health (DK43371) and The Whitaker Foundation. Offprint requests to: M. Toner     

Cell therapies for liver diseases

Cell therapies, which include bioartificial liver support and hepatocyte transplantation, have emerged as potential treatments for a variety of liver diseases. Acute liver failure, acute-on-chronic liver failure, and inherited metabolic liver diseases are examples of liver diseases that have been successfully treated with cell therapies at centers around the world. Cell therapies also have the potential to be widely applied to other liver diseases, including noninherited liver diseases and liver cancer, and to improve the success of liver transplantation. Here we briefly summarize current concepts of cell therapy for liver diseases.     

Human hepatocyte transplantation: worldwide results

The conception and animal modeling of hepatocyte transplantation along with a partial listing of human hepatocyte infusions over the last 13 years have been detailed in authoritative reviews. However, to adequately best represent the worldwide effort of moving from highly successful clinical solid liver transplants "back to" isolated hepatocyte therapy requires repeating important concepts with explanations of how or why not animal experimental data translate to human experience. This overview summarizes 78 human clinical hepatocyte transplant experiences authenticated by the authors. The human cell infusion experiences are categorized by liver disease treated (metabolic, chronic, and acute liver failure), and these are accompanied by seminal in vitro and in vivo experimental data.     

Human hepatocyte transplantation for acute liver failure: state of the art and analysis of cell sources

Liver transplantation is the only treatment available for acute liver failure. However, mortality rates remain high because of the shortage of donor organs. Indeed up to 20% of patients with acute liver failure may survive without transplantation. In the last two decades, research has focused on the development of alternative or supportive measures to deal with acute liver failure; one of the most studied is hepatocyte transplantation, because it is thought that the function of the liver can only be replaced with a biological substrate characterized by functioning liver cells. Hepatocyte transplantation has been successful in many animal models of acute liver failure, although only several clinical attempts have been made in humans with encouraging but not yet convincing results, mainly because of the lack of a reliable source of live liver cells. Allogenic and xenogenic fresh or cryopreserved hepatocytes have been tested. Recent research has focused on fetal hepatocytes and progenitor liver cells of both hepatic and bone marrow origin. The ability to preserve and bank human hepatocytes would allow pooling of cells from multiple donors to increase the numbers for transplantation. The development of a reliable and large-scale available source of live liver cells would probably have a major impact on the introduction of hepatocyte transplantation in clinical practice.     

Hepatocyte transplantation: a review of worldwide clinical developments and experiences

Hepatocyte transplantation is a promising treatment for several liver diseases and can also be used as a "bridge" to liver transplantation in cases of liver failure. Although the first animal experiments with this technique began in 1967, it was first applied in humans in 1992. Clearly, the most important advantage of this treatment, compared with liver transplantation, is its simplicity, since no surgery is required for implantation of the cells. Much work has been done over the years to maximize the number of viable hepatocytes that can be isolated from a liver, to prepare the cells prior to transplantation so that the outcome will be more successful, and to identify the optimal site for implantation. We review these efforts along with the worldwide clinical experience with hepatocyte transplantation during the last 13 years.     

An ideal liver support system and xenotransplantation

Currently, the rapid increase in the number of candidates for orthotopic liver transplantation has resulted in a shortage of donor organs and, especially for fulminant hepatic failure, an urgent need for transplantations. Thus the need for a liver support system as a "bridge" before liver transplantation is also urgent, as is the need for treatment of reversible acute liver disease. Various liver support systems have been proposed, for example, cross-circulation systems, extracorporeal liver support systems, hemodialysis, and hemadsorption, but these are not considered to function sufficiently. Demetriou's system, Sussman's system, and Gerlach's system have reached clinical trials. The ideal liver support system should have significant metabolic capacity, the ability to be used in continuous treatment, simplicity of use, biocompatibility, ready availability, consistency, economy, and safety from viral infection, but no system meets all of these criteria.     

Liver transplantation for fulminant hepatitis

Liver transplantation has been recognized as an effective therapeutic method for end-stage liver disease in Japan. Fulminant hepatic failure is also an indication for liver transplantation, and the number of patients undergoing liver transplantation has been increasing. Reversibility and urgency are characteristics of fulminant hepatitis. If given appropriate critical support, many patients recover spontaneously. However, many patients develop cerebral edema or multiorgan failure before the liver can regenerate. Indications, operative procedures, and outcome of liver transplantation for fulminant hepatitis are discussed here. At Shinshu University, 23 of 169 cases of liver transplantation were for fulminant hepatitis. One transplantation was from a cadaveric donor and 22 from living donors. The actuarial 5-year patient and graft survival rate was 85.4%. Although some problems remain in liver transplantation for fulminant hepatitis, the results are better than those of conventional therapy. Therefore patients with fulminant hepatic failure should be listed for liver transplantation when grade 2 hepatic encephalopathy develops. Moreover, in cases of severe acute hepatitis, i.e., before patients develop grade 2 encephalopathy, liver transplantation should be considered among choices of therapy in the near future.     

Fulminant hepatic failure: the role of liver transplantation as primary therapy

Fulminant hepatic failure is a clinical syndrome with a high mortality rate when traditional supportive therapy is used as treatment. Orthotopic liver transplantation has been proposed as a therapeutic option. Clinical and logistic difficulties include the rapid deterioration of the patients, unpredictable recovery, and the immediate need for a donor organs. Including this series, a total of 41 patients with fulminant hepatic failure have been transplanted, with a survival rate of 61 percent. We have reported eight liver transplantations carried out in six patients. Four of the patients survived (66 percent). Death was due to irreversible neurologic dysfunction in one patient and fungal sepsis in one patient. These results indicate that orthotopic liver transplantation is a practical therapeutic option for fulminant hepatic failure which should be considered early, before neurologic deterioration becomes irreversible.     

Development of a Bioartificial Liver Using Isolated Hepatocytes

Abstract: Severe liver disease is very often life-threatening and dramatically diminishes quality of life. Liver support systems based on detoxification alone have been proved ineffective because they cannot correct biochemical disorders. An effective artificial liver support system should be capable of carrying out the liver's essential processes, such as synthetic and metabolic functions, detoxification, and excretion. It should be capable of sustaining patients with fulminant hepatic failure, preparing patients for liver transplantation when a donor liver is not readily available (i.e., bridge to transplantation), and improving the survival and quality of life for patients for whom transplantation is not a therapeutic option. Recent advances in cell biology, tissue culture techniques, and biotechnology have led the way for the potential use of isolated hepatocytes in treating an array of liver disorders. Isolated hepatocytes may be transplanted to replace liver-specific deficiencies or as an important element of an auxiliary hybrid, bioartificial extracorporeal liver support device, which are important therapeutic applications for treating severe liver disease. Recently, several hepatocyte-based liver support systems have been proposed. Although there is no current consensus on its eventual design configuration, the hollow fiber hepatocyte bioreactor shows the greatest promise. Furthermore, application of tissue engineering technology, based on cellsurface interaction studies proposed by our group and others, has enhanced interest in the development of highly efficient hybrid, bioartificial, liver support devices.     

Extracorporeal liver perfusion system for successful hepatic support pending liver regeneration or liver transplantation: a pre-clinical controlled trial.

BACKGROUND: There is a well recognized need for a system capable of providing effective support for patients with hepatic failure pending liver regeneration or liver transplantation. Recent attempts of using bioartificial liver containing encapsulated porcine hepatocytes, the deployment of emergency whole liver, or hepatocyte transplantation are complex and not consistently successful. The technique of ex vivo hepatic perfusion developed and used clinically by Abouna in the 1970s, has now been redesigned in a perfusion circuitry that mimics the physiological conditions of a normal liver. Before clinical application of this system, a preclinical trial was carried out in dogs with induced hepatic failure. METHODS: Acute hepatic failure was induced in dogs by an end-to-side porto caval shunt, followed 24 hr later, by a 2-hr occlusion of the hepatic artery. All animals (n=18) were medically supported and were divided into three groups. In the control group (n=6) only medical support was used. In the experimental group (n=12) the animals were connected to the ex vivo liver support apparatus during acute hepatic failure via an AV shunt using a dog liver (n=6) or calf liver (n=6) (after a temporary extracorporeal bovine kidney transplant to remove preformed xeno antibody). Hepatic perfusion was carried out at 37 degrees C through the hepatic artery and portal vein at physiological pressures, and blood flow rate for 6-8 hr. RESULTS: All control animals died of progressive hepatic failure at 14-19 hr after clamping the hepatic artery. The animals treated with ex vivo liver showed remarkable clinical and biochemical improvement. Five animals survived for 36-60 hr. Another seven animals recovered completely and became long-term survivors with biochemical and histological evidence of regeneration of their own liver. Biopsy of the allogeneic ex vivo liver at the end of perfusion showed some interstitial edema. Similar biopsy of the xenogeneic calf liver showed only mild and delayed xenograft rejection, which was most likely due to removal of preformed xeno antibody by temporary transplantation of the calf kidney before liver perfusion. CONCLUSIONS: The observations and results obtained in this trial strongly confirm that extracorporeal perfusion through a whole liver, using the system described, is very successful and cost effective for the treatment of acute, but reversible hepatic failure, as well as serving as a bridge to liver transplantation. The time has come for this form of liver support technology to be reintroduced and widely used.     

Bioartificial liver support

Orthotopic liver transplantation is the only definitive therapy for patients with fulminant hepatic failure (FHF). However, due to shortage of organs, a large number of patients die before a liver can be procured for transplantation. In FHF the need for a liver is particularly urgent because of rapid deterioration in the patients' condition with the onset of cerebral edema and intracranial hypertension leading to irreversible brain damage. It is thus necessary to develop an extracorporeal liver support system to help maintain patients alive and neurologically intact until an organ becomes available for transplantation. Multiple attempts have been made, ranging from the use of plasma exchange to utilization of charcoal columns and extracorporeal devices loaded with liver tissue to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use, and FHF due to multiple causes continues to be associated with significant morbidity and mortality. In this paper, the authors review the history of extracorporeal liver support for acute liver failure and discuss their experience with a hollow fiber bioartificial liver support system utilizing porcine hepatocytes in the treatment of patients with acute liver failure. Received: July 7, 2000 / Accepted: October 12, 2000     

State of hepatocyte transplantation: a risk or a chance?

Over the past few years, hepatocyte transplantation has been considered as an alternative method for orthotopic liver transplantation for the treatment of various liver diseases. Beside curative approach for genetic metabolic deficiencies (familial hypercholesterolemia, hemophilia, etc.), it could be a useful tool for bridging the waiting period until an appropriate donor organ is obtained. In preclinical animal studies, hepatocytes injected intraperitoneally, intraportally or into the spleen settle down in the diseased liver. This enables genetic modification to correct inborn metabolic deficiencies and improves survival in acute liver failure. In 1992, the first clinical transplantation of isolated hepatocytes in 10 patients was performed. In 1998, Fox and coworkers described the successful transplantation of allogeneic liver cells in a child with Crigler-Najjar syndrome. Accomplished studies of Strom et al. resp. Bilir et al. of the same year proved the effectiveness of liver cell transplantation for transient treatment of acute liver failure. Prerequisite of this cell-based therapeutic strategy is a sufficient amount of highly differentiated hepatocytes, hence, a well established in-vitro cell-culture technique is necessary to yield a reproducible number of proliferating hepatocytes and to preserve the physiological cell function. This review discusses the different experimental approaches regarding the cultivation of human hepatocytes and also the use of alternative cell sources (like animal hepatocytes, immortalized cells of human origin, progenitor cells from fetal human liver/liver stem cells) for hepatocyte transplantation.