Key challenges to the development of extracorporeal bioartificial liver support

Hangzhou, China

Key challenges to the development of extracorporeal bioartificial liver support systems

Li-Fu Zhao, Xiao-Ping Pan and Lan-Juan Li

Hangzhou, China

BACKGROUND: For nearly three decades, extracorporeal bioartificial liver (BAL) support systems have been anticipated as promising tools for the treatment of liver failure. However, these systems are still far from clinical application. This review aimed to analyze the key challenges to the development of BALs.

DATA SOURCE: We carried out a PubMed search of Englishlanguage articles relevant to extracorporeal BAL support systems and liver failure.

RESULTS: Extracorporeal BALs face a series of challenges. First, an appropriate cell source for BAL is not readily available. Second, existing bioreactors do not providein vivolike oxygenation and bile secretion. Third, emergency needs cannot be met by current BALs. Finally, the effectiveness of BALs, either in animals or in patients, has been difficult to document.

CONCLUSIONS: Extracorporeal BAL support systems are mainly challenged by incompetent cell sources and flawed bioreactors. To advance this technology, future research is needed to provide more insights into interpreting the conditions for hepatocyte differentiation and liver microstructure formation.

(Hepatobiliary Pancreat Dis Int 2012;11:243-249)

bioartificial liver; liver failure; cell source; bioreactor

Introduction

Liver failure is the inability of the liver to perform its normal detoxification, biosynthesis, and/ or biotransformation functions. The clinical presentation of liver failure includes a prolonged prothrombin time, encephalopathy, and jaundice. Regardless of the etiology, liver failure can be divided into two categories: acute (ALF) or acute-on-chronic (AoCLF).[1]Both are accompanied by high mortality.[2-4]Transplantation is still the only ultimate solution for end-stage liver failure, but its application is hampered by a world-wide scarcity of donor organs. In this context, extracorporeal liver support systems have been expected to provide a bridge to transplantation or to provide an opportunity for the native liver to regenerate.[5,6]

Depending on whether they are loaded with metabolically active hepatocytes or not, these systems can be roughly classified into two types: artificial or bioartificial liver (BAL) systems. It is widely accepted that an artificial liver, which can only detoxify, is insufficient to support liver failure patients, while in theory an ideal hepatocyte-based BAL could provide most or even all normal liver functions.[6-15]

However, it has to be recognized that the BAL is still far from being ready for routine clinical application. BAL systems currently under clinical trials include ELAD,[16-20]HepatAssist,[21-26]BLSS,[27-29]AMC-BAL,[30-32]MELS,[33-35]RFB,[36,37]and HBAL/TECA-HALSS.[38,39](Table 1). Among these, the HepatAssist system was the first and initially reported in the 1980s.[23]All of these systems were found to be safe in phase I clinical trials (Table 2). However, to date, only two randomized controlled clinical trials exploring the effectiveness of BALs have been reported,[18,24]and the results were not encouraging, suggesting that the development of an effective BAL system with widespread clinical acceptance must be quite difficult. Hence we aimed to analyze the key issues restricting the developmentof extracorporeal BAL support systems. Detailed descriptions of individual research contents will not appear in this review.

Table 1. Characteristics of BAL systems currently under clinical trials

Table 2. Clinical efficacy and adverse events of BAL systems under phase I trials and randomized controlled trials (RCTs)

Absence of an ideal cell source for BAL support

Cell sources that have been previously used in extracorporeal BAL treatment in patients and/or large animal models include primary pig hepatocytes, primary human hepatocytes, and human liver tumor-derived cell lines. Primary pig hepatocytes are the biological components of all of the BALs currently under clinical trials, except for ELAD. Consequently, zoonosis and immunogenicity restrict their widespread use. In addition, although a high degree of metabolic similarity is found between human and pig hepatocytes,[40]the latter are unable to synthesize coagulation factors that function in the human body.[41]

Primary human hepatocytes have only been used in three clinical trials, based on ELAD or MELS. There are two single-case studies and one phase I clinical trial.[20,34,35]Common applications of primary human hepatocytes are very difficult because both yield and quality are poor. For one thing, healthy donor livers are scarce, so that only organs or tissues discarded at transplantation (i.e. with fibrosis and steatosis) are available for BALs. For another, primary human hepatocytes do not proliferate efficientlyin vitroand demonstrate a serious loss of viability after the freeze-thaw process.

Among non-primary cell sources, only the C3A cell line, a HepG2 hepatoma subclone, has been used in clinical trials and was adopted for the ELAD system.[16-19]Unfortunately, no improvement in either survival or biochemical parameters was demonstrated in a pilotcontrolled clinical trial.[18]Several other human liver tumor-derived cell lines, such as GS-HepG2 (glutamine synthetase, GS), HepG2-GS-3A4, and FLC-4, were used in BAL support in large animal models.[42-46]Prolongations of survival, with or without statistical significance, were achieved in these studies. However, none have been so far applied in clinical trials. Poor differentiation and the potential risk of metastatic tumor formation might be the main hurdles.[40,47]In addition, a hepatocyte line with high GS expression, for ammonia removal, would potentially increase the production of glutamine. This may further disturb brain function in patients with liver failure.[48]

Cell sources that have not yet been tested in extracorporeal BAL systems include immortalized fetal human hepatocytes, immortalized adult human hepatocytes, and human stem cell-derived hepatocytes. More than a decade ago, some researchers claimed that their immortalized fetal or adult hepatocyte lines were promising cell sources for BALs.[49-52]However, the followup research and applications are still absent, which may suggest that they encountered insurmountable difficulties. Recently, a new immortalized human fetal hepatocyte line, cBAL111, was established by overexpression of the reverse transcriptase of telomerase (hTERT).[53]However, this cell line fell under scrutiny because it was found to have considerable variations at the genetic level, compared with primary hepatocytes in BALsin vitro.[54]

The method of reversible immortalization was once encouraging. In this process, the immortalizing genes, i.e., simian virus 40 large T antigen (SV40LT) or hTERT, can be excised using a Cre/LoxP site-specific recombination. Then, an increase of liver-specific functionality can be shown later.[55-58]However, it was reported that even the reversibly immortalized human hepatocyte line, NKNT-3,[57]was poorly differentiatedin vitroafter reversion.[53]Another reversibly immortalized human hepatocyte line, 16-T3,[58]has never been compared with mature human hepatocytes at the genetic level. Although stem cells from different tissues have the potential to differentiate into hepatocyte-like cells, some issues, such as insufficient quantity, incomplete functionality, ethical controversy, and safety still challenge the clinical availability of these cells.[59,60]

From all of this information, a relevant conclusion can be drawn. As the biological component of extracorporeal BAL support systems, an appropriate cell source should combine the following characteristics: (i) nearly full functionality of mature human hepatocytes, (ii) unlimited life-span and proliferative capacityin vitro, and (iii) no potential risk of metastatic tumor formation, zoonotic transmission, or immunogenicity. Unfortunately, no such cell source has yet been found. Some thought that a highly differentiated human hepatocyte line was most likely to be competent in BALs.[15]Others, however, argued that it was difficult to replace liver functions with a single cell line.[61,62]The liver is formed by hepatocytes and a variety of non-parenchymal cells such as Kupffer cells, sinusoidal endothelial cells and stellate cells. These cells communicate with each other and maintain the physiological functions of the liver. Coculture techniques, therefore, are considered promising for obtaining cell sources for BALsin vitro.[62]While it is possible to obtain an ideal cell source ultimately, the existing bioreactor design remains flawed.

An ideal BAL bioreactor should also provide anin vivo-like environment, where the viability and functionality of a large number of hepatocytes can be optimally maintainedin vitro. However, such highlevel simulation has not been achieved, as oxygenationand bile secretion are two major on-going controversial issues.

In vivo-like oxygen supply

The human liver has a dual blood supply; it is fed by the hepatic arteries and the portal vein. Both sources enter the sinusoids and provide oxygen to hepatocytes. The hepatic blood flow rate in physiological conditions averages 1450 mL/min, which is roughly equivalent to a quarter of resting cardiac output.[63]It is not difficult to infer that an adequate and controllable oxygen supply is the prerequisite for the maintenance of hepatocytes on a large scale. In most BALs currently under clinical trials, the perfusion fluids are plasma or blood ultrafiltrate, since whole blood perfusion inevitably leads to hemolysis and coagulation. In contrast to whole blood, which contains red blood cells, the oxygencarrying capacity of plasma or blood ultrafiltrate is quite insufficient. Therefore, hepatocytes in BAL bioreactors are inevitably exposed to hypoxia.[64,65]Two strategies have been used to solve this problem so far.

One solution is the application of an integral oxygenator, as in the AMC-BAL and MELS systems.[31,34]In these systems, the oxygenation capillaries are uniformly distributed throughout the bioreactor cavity or interwoven with the cell attachment matrix. Such a structure enables hepatocytes to acquire oxygen from its immediate surroundings. An integral oxygenator may be superior to an external one, but neither has the self-regulation ability possessed by organisms. In physiological environments, hemoglobin acquires oxygen molecules at high partial pressures of oxygen (pO2), and releases them at low levels of pO2. The oxygen affinity of hemoglobin is affected by several factors, such as body temperature, carbon dioxide concentration, and pH. Thus, organisms can protect themselves from hypoxia and oxygen toxicity by relying on self-regulation, whereas these artificial oxygenators cannot.

Another strategy is the supplementation of perfusion fluids with red blood cell substitutes (e.g., artificial oxygen carriers), such as perfluorocarbons (PFCs) and/ or hemoglobin-based O2carriers (HBOCs).[66-68]PFCs are capable of carrying large quantities of oxygen to tissues,[69]but can reach levels that are toxic. Moreover, phase III clinical trials showed that an increased risk of stroke is associated with PFCs.[70]At least to some extent, HBOCs mimic thein vivooxygen supply but, unfortunately, a meta-analysis showed a significant increase in the risk of death and myocardial infarction by HBOCs.[71]The serious adverse side-effects of PFCs and HBOCs make their future very uncertain.

Absence of bile secretory function

No existing BAL bioreactor has a biliary system capable of collecting bile produced by hepatocytes and moving it out of extracorporeal circulation. To solve this, a combination of a BAL bioreactor and an artificial liver device, such as albumin dialysis, carbon absorption, or plasma exchange, has been proposed.[34,38,39]However, this strategy may be questionable. First, it is not clear what proportion of bile is retained intracellularly, and whether it can be washed away by an artificial liver device. Second, these combinatorial devices make it difficult to discern whether it is the artificial liver device or the BAL bioreactor that contributes to the efficacy.

Lack of convenience

To serve as a piece of emergency equipment, a BAL should be easy to operate and immediately available at any time. However, before a BAL system can be connected to a live body, operators must complete a series of procedures. First, for a pig hepatocyte-based BAL, complications related to sacrificing animals and isolating cells must be overcome. Second, for a primary human hepatocyte-based BAL, human organ tissues must be collected and/or thawed, and hepatocytes must then be isolated. In addition, for a hepatocyte line-based BAL, there needs to be a large-scale cultivation and/or freeze-thaw process. And finally, there needs to be a cell adhesion process within the bioreactor. All of these obstacles are complicated and time-consuming.

Difficulty of efficacy evaluation

In animal experiments, evaluation of a BAL system loaded with human-derived hepatocytes is not easy since the metabolic capability of human cells still differs in some aspects from that of pig hepatocytes.[41]The difference between human and other species is even greater.[40]The clinical efficacy of BALs is also difficult to show since, in many cases, BAL therapy just acts as a bridge, with the ultimate solution being liver transplantation.

Conclusions

For nearly 30 years, extracorporeal BAL support systems have raised great expectations for the treatment of liver failure. However, so far, none of these systems is ready for routine clinical use. BAL systems experience bottlenecks in several areas, including cell sourcing, bioreactor design, convenience, and efficacy assessment. To makethis technology more hopeful, two things are essential: 1) we need a highly differentiated human hepatocyte line; and 2) we need a bioreactor capable of providing anin vivo-like environment for cells. Future research should not only be focused on better understanding hepatocyte proliferation and differentiation, but also on studying liver microstructure formation, such as liver microvessels and bile canaliculi.

Contributors: ZLF wrote the main body of the article. PXP provided advice. All authors contributed to the design and interpretation of the study and to further drafts. LLJ is the guarantor.

Funding: This work was supported by grants from the National Natural Science Foundation of China (30630023) and the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (81121002).

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Accepted after revision March 7, 2012

It requires wisdom to understand wisdom; the music is nothing if the audience is deaf.

—Walter Lippmann

December 13, 2011

Author Affiliations: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Zhao LF, Pan XP and Li LJ)

Lan-Juan Li, MD, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571-87236759; Fax: 86-571-87236759; Email: ljli@zju.edu.cn)

© 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(12)60155-6