Artificial liver support

Norman L. Sussman, MD
James H. Kelly, PhD

Clin Invest Med 1996; 19 (5): 393-99.

[résumé]

Drs. Sussman and Kelly are from Baylor College of Medicine and Amphioxus Inc., Houston, Tex.

Paper reprints may be obtained from: Dr. Norman L. Sussman, 5250 Ariel St., Houston TX 77096; budsus@watanabe.com

Contents

Abstract

Without adequate liver function, the body is unable to sustain several vital metabolic functions, such as energy supply, acid-base balance and thermoregulation. Whereas the clinical picture of chronic liver failure is often dominated by portal hypertension, fulminant hepatic failure (FHF) is typically characterized by an acute metabolic deficit. Another important distinction between these two conditions is that the liver can recover from an acute injury such as FHF. Hepatocytes retain the ability to divide in vivo; therefore, recovery from FHF is possible, although rare, if the liver can regenerate before the patient succumbs to the disease. This review examines the theoretical and practical aspects of metabolic liver support, with FHF as the paradigm.

Résumé

Sans une fonction hépatique adéquate, l'organisme est incapable de maintenir plusieurs fonctions métaboliques vitales, telles que l'apport énergétique, l'équilibre acide-base et la thermorégulation. Tandis que les manifestations cliniques de l'insuffisance hépatique chronique sont souvent dominées par l'hypertension portale, l'insuffisance hépatique fulminante (IHF) est plutôt caractérisée par un déficit métabolique aigu. Une autre distinction importante entre ces deux états est que le foie peut récupérer d'une lésion aigüe comme l'IHF. Les hépatocytes conservent la capacité de se diviser in vivo. Bien que rare, la récupération d'une IHF est donc possible, à condition que le foie puisse se regénérer avant le décès. Cet article révise les aspects théoriques et pratiques du soutien métabolique hépatique, en utilisant l'IHF comme paradigme.

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Introduction

Fulminant hepatic failure (FHF) is defined as rapidly progressive liver dysfunction that leads to encephalopathy within 8 weeks of the onset of symptoms1 or within 2 weeks of the onset of jaundice.[2] The features common to both definitions are a disturbance of consciousness and an absence of pre-existing liver disease. The neurologic disturbance indicates that the liver is functioning below a critical mass (probably 25% to 35% of normal, although the exact levels are not known). The absence of pre-existing liver disease indicates that the mechanism of injury can be reversed and that the liver is capable of full recovery.

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Liver regeneration

The regenerative capacity of the liver is enormous and is the foundation of recovery from FHF. Studies involving rats demonstrate that almost every hepatocyte undergoes a round of cell division about 20 hours after partial hepatectomy.[3,4] Subsequent rounds of cell division are not as well coordinated, but the liver can double its hepatocyte mass in less than 1 day, which is critical in the treatment of FHF. Even patients with advanced disease may recover rapidly once regeneration begins,[5,6] and those who recover have no residual liver disease and a normal life expectancy.[7]

The critical task is to distinguish between patients who will recover spontaneously and those who will not. If recovery is unlikely, emergency orthotopic liver transplantation should be performed.[8,9] This is a drastic and expensive form of therapy but a justifiable one in the context of this disease; spontaneous recovery from FHF occurs in about 25% of cases[2] whereas the rate of survival after liver transplantation is about 65%.[10] This therapeutic option creates a dilemma: transplantation performed early in the course of FHF may have a success rate of more than 90%,[11,12] but transplantation is needless for some patients. Surgery performed late in the course of FHF is often unsuccessful, and deciding on transplantation later in the course of the disease reduces the chance of finding an organ in time.

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Liver-assist devices

To be useful, a liver-assist device must bring the body's net liver function above a critical threshold, generally believed to be about 30% of normal function. In simple terms, the functional hepatocyte mass available to an adult patient must be approximately 400 g to maintain critical body functions. If this mass is achieved, obvious advantages are anticipated. The complications of liver failure would be alleviated, dependent organs would continue to function normally and metabolic stability would promote earlier liver regeneration. All of these factors are expected to contribute to a shorter hospital stay and reduced costs as well as better use of donated livers. There are severe limits on the number of livers available for transplantation; livers that are not used in transplantation to treat FHF can be used more appropriately in transplantation in patients with irreversible diseases.

Reports of liver-assist devices first appeared in the 1950s, and various forms have been tested since then. Two approaches, mechanical and biochemical, have been described. The history of liver-assist devices has recently been detailed.[13]

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Mechanical devices

Without normal liver function, metabolic byproducts begin to accumulate. These byproducts may play a role in the secondary organ failures that characterize FHF; for example, accumulated benzodiazepine-like substances may be related to hepatic encephalopathy.[14] Evidence suggests that the build-up of these metabolic byproducts suppresses cell regeneration and that the byproducts can be removed by charcoal adsorption.[15] The use of charcoal and various exchange resins has therefore been proposed as a method of improving the environment for hepatocyte regeneration.

The problem with mechanical devices is that they are metabolically inert. The liver's function is more biochemical than excretory. Mechanical devices cannot perform intermediary metabolism and do not synthesize liver-specific products; therefore, homeostasis is not restored until the native liver regenerates or recovers a substantial part of its function. Thus, use of these devices may improve mental status,[16] but they do not promote liver regeneration and their use has not altered the death rate in patients with FHF.[17]

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Bioartificial devices

An appreciation of the need for a biochemical approach to liver support was eloquently stated by Eiseman and associates in 1965.[18]

The metabolic complexity of the liver makes it unlikely that attempts to eradicate only a single "toxic" product of liver failure would meet with much lasting clinical success. In all likelihood, only another normal liver can adequately substitute for one that has failed.

The success of liver transplantation in the treatment of FHF has vindicated this view, and a recent report confirms that liver function can be provided by extracorporeal liver perfusion.[19] The problem with this strategy is the unavailability of organs. Animal livers function for a very short time when perfused with human blood,[13] and human livers are a scarce resource. These limitations have necessitated the development of hybrid devices that combine the effectiveness of a normal liver with the convenience of a medical device; these are referred to as bioartificial devices.

Biologically active devices have a support structure that houses a mass of hepatocytes and a mechanism for continuous perfusion by plasma or blood. The various prototypes currently under development differ in the geometric aspects of the support structure, the nature of the perfusate and the source of hepatocytes.

Geometric aspects

Most artificial livers involve some variant of the hollow-fibre cartridge (Fig. 1[20]). This cartridge, similar to a hemodialysis device, contains numerous hollow fibres composed of a semipermeable material. The ends of the fibres are embedded in an epoxy resin so that the device has two compartments, an intracapillary space (ICS) and an extracapillary space (ECS). In a typical arrangement, cells are located in the ECS and blood or plasma is pumped through the ICS. This arrangement is thought to reduce turbulent blood flow and hence to reduce clotting. There is no other theoretical objection to the opposite orientation, and Nyberg and associates[21] have reported on a device in which cells are embedded in a protein matrix in the ICS.

Perfusate

Devices may be perfused with plasma or with whole blood. Plasma perfusion has two advantages: plasma is less likely than blood to clot during treatment, and it can be filtered before its return to the blood stream (eliminating cell contamination). On the other hand, the circuit is considerably more complicated, and the fluid has a markedly lower oxygen-carrying capacity than blood. Perfusion with whole blood is simpler and provides the oxygenation necessary to sustain very high-density cultures. In our experiments, blood flow rates of 75 to 100 mL per minute are sufficient to maintain cartridge oxygenation.

Hepatocytes

To be effective, cell-based therapy must supplement existing liver function enough to satisfy the metabolic needs of the patient. The adult liver weighs about 1500 g and contains approximately 1200 g of hepatocytes. Hepatic encephalopathy occurs when liver function falls below 25% to 35% of normal, i.e., when less than 300 to 420 g of viable hepatocytes remain. Therefore, a device that provides the functional equivalent of several hundred grams of liver should be able to assist the existing liver.

Primary hepatocytes

Most of the current work in cell-based liver therapy involves the use of primary hepatocytes, cells isolated from fresh liver and maintained in culture.[22­24] The main drawback to their use is that they do not divide in vitro. A steady supply of fresh cells is required, and cell isolation is technically challenging and time consuming. Hence, the time required to prepare devices may be limiting. Rozga and associates[25] have developed several techniques pertinent to this field. With these techniques, cells can be isolated efficiently and attached to microcarrier beads. Cells treated in this manner can be frozen, stored and thawed when needed. Animal studies in which 10% of the animals' hepatocyte mass was isolated suggest that this method is efficacious,[25] and a dramatic result from the use of a hybrid device in series with a charcoal column has been reported.[26]

Immortalized liver cells

An immortalized cell line is an attractive alternative to primary hepatocytes; the cells appear to function quite well,[27] and cell division is practically unlimited. A human line is even more attractive; it obviates concerns about species-specific metabolic differences, and the infusion of human proteins is less likely to cause immune-mediated sequelae, especially after prolonged or repeated use. We have developed an extracorporeal liver-assist device (ELAD), which consists of a hollow-fibre cartridge populated with an immortalized liver cell line. The cells (C3A) express normal liver-specific metabolic pathways such as ureogenesis, gluconeogenesis and P-450 activity, and they secrete clotting factors and other liver-specific proteins (Dr. James H. Kelly, Amphioxus Inc, Houston, Tex: unpublished observations, 1989). Like normal liver cells, they are strongly contact-inhibited, i.e., they cease dividing when their culture space is filled. Extensive testing has failed to yield any infectious or adventitious agents.

These devices are manufactured by injecting 2 to 5 g of C3A cells into the extracapillary space of a hollow-fibre cartridge. The cells grow to confluence for 3 to 4 weeks, and then cease dividing. Once confluent, the cultures remain stable indefinitely, and we have seen no deterioration in function after 6 to 8 months. In-vitro and in-vivo assays of function have consistently demonstrated that each cartridge has a metabolic capacity of about 200 g of normal liver.

The obvious question is whether patients treated with the use of these cells are at risk of a tumour. The risk is probably very low for three reasons. First, C3A cells express normal surface antigens and are therefore subject to host immune defences. Second, blood does not normally come into contact with the cells, and, third, the treatment system is designed to prevent an infusion of C3A cells if a fibre ruptures.[28] In the final analysis, the risk-to-benefit ratio is low enough to warrant the use of this device in the management of FHF. Long-term follow-up is needed to confirm whether the tumour risk is real concern. A comparison of the advantages and disadvantages of the two sources of liver cells is shown in Table 1.

Testing of artificial livers in animals

Table 2 summarizes published articles concerning tests of these devices in animals.

Primary hepatocytes

Rozga and associates[25] tested their bioartificial liver in an ischemic model of liver failure. Animals treated for 6 hours were hemodynamically stable and levels of blood chemicals were improved significantly (higher glucose level and lower ammonia and lactate levels). Jauregui and associates[29] conducted studies of galactosamine-induced liver failure in rabbits and demonstrated delayed onset of encephalopathy and improved survival after 80 hours.

C3A cells

We have tested the ELAD in an acetaminophen model of FHF.[30] Animals can be sustained during the nadir of liver disease and can recover completely, with liver regeneration in 42 to 48 hours.[21,31]

Human testing of artificial livers

Rozga and associates[26] have tested their device in more than 20 patients. A dramatic fall in intracranial pressure was reported in one case. A series of seven patients was recently published.[32] We have tested our device in 11 patients in the United States[33­35] and in 12 patients at King's College Hospital in London, England.[36] A summary of patient trials is shown in Table 3.

The preliminary data from the use of both devices are exciting, but it would be unwise to make any claims about effectiveness until multicentre randomized controlled trials have been performed. At the time of writing, both groups are planning such trials.

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The future

Metabolically active liver-assist devices are a feasible and logical method of sustaining patients with FHF. If we can replace liver function with an external device, metabolic instability and multisystem failure can be prevented. The most challenging of medical emergencies may become an easily treatable condition, similar in complexity to the treatment of acute tubular necrosis with hemodialysis, with most patients recovering fully in a few days.

If these predictions prove to be correct, they will have a significant effect on the health care system. A reduction in secondary organ failure will reduce the need for intensive care, and it may ultimately be possible to treat patients outside the intensive care unit. Patients who are stable before liver transplantation have better survival rates, less frequent graft failure and earlier hospital discharge. Patients who recover without the need for transplantation are even more fortunate; they are spared the risk and expense of surgery as well as a lifetime of immunosuppression and medical care. In addition, the organs can be put to better use; by avoiding the needless liver transplantation in patients with FHF, we could make about 300 livers annually available in the United States for patients with irreversible liver disease.

Although these comments have been restricted to FHF, other uses for liver support are envisioned. Primary graft nonfunction after transplantation is an extension of the FHF theme; it may be possible to recover graft function without a second transplantation. In cases of chronic liver disease, acute decompensation before transplantation has a deleterious effect on survival. Improved liver function is likely to reduce morbidity and mortality after surgery. Similarly, liver support for patients with cirrhosis during nonhepatic surgery is likely to improve survival in this high-risk group. Finally, some patients with end-stage liver disease may benefit from long-term, intermittent ELAD therapy, in a situation analogous to long-term hemodialysis in end-stage renal disease. Although many hurdles remain before end-stage liver disease is treated with long-term "liver dialysis," we may eventually be able to make the same choices in the treatment of liver disease as we now make in the treatment of renal disease.

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Acknowledgements

This work was supported in part by a grant from the National Institutes of Health (DK46228) to Dr. Kelly. The publication of this article was supported in part by a grant from the Fonds de la recherche en santé du Québec.

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CIM: October 1996 / MCE: octobre 1996
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