Configuration of a new bioartificial liver support system and in vitro evaluation of its functions

The aim of this study was to configure a new bioartificial liver (BAL) support system and evaluate its functions in vitro. Chinese experimental miniature pig hepatocytes were isolated by an in situ recirculating collagenase perfusion method and were cultured in serum-free medium with restriction attachment and spinner technique to form hepatocyte spheroid suspensions containing 1.0 x 10(10) hepatocytes. The BAL support system was configured by inoculating the hepatocyte spheroids into the cell circuit of a hollow fiber bioreactor (BIOLIV A3A, Cell Biotech Limited, HK, China). The number and viability of hepatocytes, the levels of alanine aminotransferase (ALT), total bilirubin (TBi), and albumin (ALB) in the circulating hepatocyte suspension and RPMI-1640 medium, and lidocaine metabolism were determined during 6 hr of circulation in the BAL devices. Independent experiments were performed 5 times. There were no significant changes in the number and viability of the hepatocytes during the circulation period. The BAL support system demonstrated substantial albumin synthesis and lidocaine metabolism. The results indicate that the new BAL support system has the ability to perform liver functions and could be used to treat liver failure or provide temporary liver support in patients who are candidates for liver transplantation.     

新型人源细胞混合型生物人工肝的安全性

背景：目前原位肝移植是惟一有效治疗急性肝衰竭的方法,但其存在供体匮乏的问题。人工肝可作为肝移植过渡支持手段。 目的：观察新型人源细胞混合型生物人工肝的安全性。 方法:在全接触灌流型生物反应器接种微载体微重力中国人肝细胞系1细胞,结合血浆灌流对D-氨基半乳糖诱发急性肝功能衰竭模型食蟹猴进行治疗,治疗过程中观察循环管路的密闭性、不良反应及监测动物的生命体征变化,治疗结束后取灌流型生物反应器中的中国人肝细胞系1细胞,把细胞碎片与细胞分别接种至裸鼠的颈部、后背部皮下进行比较。 结果与结论：对急性肝功能衰竭食蟹猴模型的治疗过程中未发现循环管路中有液体渗漏,未发生出血、过敏、高热及其他严重的不良反应,生命体征平稳。接种后4周时,细胞碎片接种裸鼠未见接种部位出现种植瘤,细胞接种裸鼠共有10个接种部位出现肿瘤,致瘤率为100%。说明新型人源细胞混合型生物人工肝是安全可靠的,可进一步探讨其有效性,有望用于各种动物实验研究及各期临床实验研究。     

In vitro evaluation method of bioartificial liver function: constant infusion test

 Various types of bioartificial livers (BALs), which are extracorporeal medical devices incorporating living hepatocytes in cartridges, have been developed. However, it is difficult to compare metabolic functions among BAL types or to know what proportion of the normal liver functions could be replaced by a BAL, because there is not a well-established method for the quantitative evaluation of BAL functions. In our series of studies, we have proposed methods for performing drug-loading tests and procedures to analyze drug concentration changes for the quantitative evaluation and expression of BAL metabolic functions. In this study, constant infusion tests of lidocaine were performed on a BAL device developed in our laboratory, and lidocaine concentration changes in the perfusion medium were analyzed by using pharmacokinetic equations. The lidocaine clearance value of the BAL was precisely determined by a constant infusion test, demonstrating the usefulness of the constant infusion test for quantitative evaluation of BAL functions. Received: February 12, 2002 / Accepted: September 10, 2002 Acknowledgments This work was supported by grant JSPS – RFTF 96I 00204 from the Japan Society for the Promotion of Science and by the New Energy and Industrial Technology Development Organization. Correspondence to:H. Iwata     

Transmission electron microscopic study of hepatocytes in bioartificial liver

A bioartificial liver (BAL) was prepared by simple inoculation of hepatocytes into the inner space of hollow fibers of a hemodialyzer and it was maintained in a closed circuit for in vitro culture. Morphology of hepatocytes in the hollow fibers was studied in detail using transmission electron microscopy (TEM). The hepatocytes formed three-dimensional, rod-shaped aggregates of 200 microm in diameter throughout the whole dimension of the hollow fibers after 1 day of culture. Approximately five hepatocyte layers existed from the surface to the center of the aggregate. The hepatocytes in the aggregate displayed mostly polygonal shapes and were surrounded by five to six cells. Abundant bile canaliculi were formed between the hepatocytes and were sealed by tight junctions. The distance between the adjacent hepatocytes except the bile canaliculus domain was approximately 20 nm, and interdigitation was observed between some hepatocytes. These observations indicate that the hepatocytes formed functionally associated aggregates, that is, organoids. Although the cells facing the inner surface of the hollow fiber lost their polygonal shape and became flattened during the following several-day culture, no drastic change was observed in the morphology of the hepatocytes located inside the aggregate. After 14 days of culture, the number of living cells decreased and most of these had a deformed nucleus, few numbers of organelles, and intermittent lipid droplets.     

Hybrid bioartificial liver: establishing a reversibly immortalized human hepatocyte line and developing a bioartificial liver for practical use

Recently, much attention has been attracted by a novel therapy for liver failure using a hybrid bioartificial liver (BAL) support device that incorporates living liver cells. Researchers in various fields have considered the following cells for potential use in BALs: human embryonic stem (ES) cells; somatic stem cells; differentiated tissue cells; and cells derived from tissues of different animal species, particularly from the pig. With their pluripotency, human ES cells are extremely useful, and many research groups are joining the race to develop BALs. One such effort involves the breeding of transgenic pigs to overcome interspecies barriers. Recent reports suggest, however, that porcine endogenous retrovirus may infect human tissues, and clinical application of transgenic pigs has become a controversial issue. To avoid such risks, we recommend that researchers adopt a reversible immortalization system that uses the Cre-loxP site-specific recombination reaction targeting human hepatocytes in their final differentiated state. This system has allowed us to establish a safe human hepatocyte line that is capable of differentiation at low cost and on a large scale. We are also designing and developing an artificial liver module made of a combination of hollow fibers and nonwoven fabrics. The objective of this review article is to report our therapeutic strategy, which aims at the earliest possible introduction of the treatment of liver failure using BALs.     

Development of a new bioartificial liver using a porcine autologous biomatrix as hepatocyte support

Long-term maintenance of hepatocyte viability and differentiated function expression is crucial for bioartificial liver support. The maintenance of hepatocyte function in a bioreactor is still a problem. A major advance was the recognition that hepatocytes in attachment cultures can maintain their differentiation longer. To restore hepatocyte polarity and prolong their function, we developed a new bioreactor with a cross-flow geometry configuration and an original hepatocyte extracellular autologous biomatrix (Porcine Bio-Matrix) support. To test this new bioreactor, we compared it with a standard bioartificial liver cartridge in a suitable surgical model of acute liver failure in pigs. In our model, we performed a total hepatectomy, followed by partial liver transplantation after an 18 hour anhepatic phase. The results showed that the bioreactor containing the biomatrix was able to bridge the animal to transplantation and to sustain the transplanted liver until all function recovered (80% of animals survived, p = 0.0027). No animal survived more than 24 hours after liver transplantation in the group treated with the traditional bioartificial liver, whereas hepatocyte viability on the Porcine Bio-Matrix was 65% after 12 hours of treatment. The results suggest that our biomatrix is a suitable cell support and guarantees long-term maintenance of metabolic activity of hepatocytes. Further studies are needed, but the results obtained with this new three-dimensional bioreactor are promising, and its potential is attractive.     

In vitro comparison of two bioartificial liver support systems: MELS CellModule and AMC-BAL

Clinically applied bioartificial liver (BAL) support systems are difficult to compare with regard to overall hepatocyte-specific function and clinical outcome. We compared two clinically applied BAL systems, the Modular Extracorporeal Liver Support (MELS) CellModule and the AMC-bioartificial liver (AMC-BAL) in an in vitro set-up. Both BAL systems were loaded with 10 billion freshly isolated porcine hepatocytes, cultured for 7 days and tested on days 1, 2, 4 and 7. Average decrease in hepatocyte-specific functions over 7 days was 9.7%. Three parameters differed between both bioreactors: lidocaine elimination at days 1 and 2 was significantly higher in the AMCBAL, ammonia elimination showed a significantly higher trend for the AMC-BAL over 7 days and LDH release was significantly lower at day 7 for the MELS CellModule. In conclusion, this first in vitro comparison of two clinically applied BAL systems shows comparable functional capacity over a period of 7 days.     

Sustaining a bioartificial liver under hypothermic conditions

Bioartificial liver (BAL) devices employing xenogeneic hepatocytes are being developed as a temporary support of liver failure. For clinical applications, transporting such a device from the manufacturing site to the hospital is necessary. We investigated the effect of hypothermic treatment on the performance of the collagen-entrapment BAL device developed at the University of Minnesota. A number of chemical protectants were examined for their effectiveness in minimizing damage to hepatocytes. Preincubation with protectant (tauroursodeoxycholic acid, TUDCA) before hypothermic treatment improved posttreatment BAL performance. Oxygen consumption and albumin and urea synthesis all resumed at levels comparable to pretreatment levels. The method described will facilitate the application of BAL in the treatment of liver failure.     

Effects of anticoagulants on porcine hepatocytes in vitro: implications in the porcine hepatocyte-based bioartificial liver.

For the clinical treatment with porcine hepatocyte-based bioartificial liver (BAL), the use of an anticoagulant in the extracorporeal system is essential. In this experiment, we studied the effect of various anticoagulants on cultured porcine hepatocytes. Porcine hepatocytes were isolated and seeded at a density of 2 x 10(5) cells on a collagen-coated plate in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Twenty-four hours later, the medium was changed to DMEM with various anticoagulants such as nafamostat mesilate (NM), sodium heparin (SH) and sodium citrate (SC) at concentration used clinically. As a control, the hepatocytes were cultured in only DMEM. After culturing for 6 hours, the viability of the porcine hepatocytes, lactate dehydrogenase (LDH) release, lidocaine clearance (cytochrome p450 function) and albumin synthesis were investigated. SC did not affect either the viability or the p450 function of the hepatocytes. In the NM group, the viability of porcine hepatocytes and lidocaine clearance were decreased significantly more than in the other groups. SH did not affect the viability of porcine hepatocytes, however, it seemed to reduce the p450 function. In conclusion, SC may therefore be the optimal anticoagulant available for hepatocyte-based BAL circuit in terms of its cell toxicity.     

New advances in MR-compatible bioartificial liver

MR-compatible bioartificial liver (BAL) studies have been performed for 30 years and are reviewed. There are two types of study: (i) metabolism and drug studies using multinuclear MRS; primarily short-term (< 8 h) studies; (ii) the use of multinuclear MRS and MRI to noninvasively define the features and functions of BAL systems for long-term liver tissue engineering. In the latter, these systems often undergo not only modification of the perfusion system, but also the construction of MR radiofrequency probes around the bioreactor. We present novel MR-compatible BALs and the use of multinuclear MRS ((13)C, (19)F, (31)P) for the noninvasive monitoring of their growth, metabolism and viability, as well as (1)H MRI methods for the determination of flow profiles, diffusion, cell distribution, quality assurance and bioreactor integrity. Finally, a simple flexible coil design and circuit, and life support system, are described that can make almost any BAL MR-compatible.     

Three-dimensional culture of porcine fetal liver cells for a bioartificial liver

A three-dimensional (3-D) culture experiment of porcine fetal liver cells (FLCs) was performed using a porous resin substrate, for the purpose of developing a bioartificial liver. A long-term 3-D culture and monolayer culture as the control were performed for more than 1 month. To promote cell growth and maturation, human oncostatin M (OSM), the human leukemia inhibitory factor (LIF), or cortisol was added to the cultures, and the effect of each agent on cell proliferation and liver-specific cellular functions was investigated. The cell numbers in both the monolayer and 3-D cultures increased gradually with time, irrespective of the supplementation of the stimulating agents. In the monolayer culture, the albumin secretion of FLCs decreased rapidly, and scarce activity was detected from 2 weeks onward under all culture conditions tested. In the 3-D cultures, neither human OSM nor human LIF had any definite effect on the albumin secretion of FLCs. However, in the cultures with cortisol, albumin secretion was maintained for a considerably long period. These findings suggest that a bioartificial liver can be developed by culturing porcine FLCs with cortisol as the stimulant.     

Perifusion model for detoxification of drugs in a bioartificial liver

Detoxification of a drug in a bioartificial liver (BAL) during an in vitro experiment was theoretically carried out based on a perifusion model. The detoxification capacity assay, the rates of disappearance of the chemicals such as flow-limited and enzyme-limited drugs in the BAL system could be defined from models of hepatic perfusion-elimination relationships. When the flow-limited drug administrated under a quasi-equilibrium condition, a two-compartment model for the concentration behavior of the drug was introduced and compared with a one-compartment model. For both models, equations involving hepatic drug clearance and various pharmacokinetic parameters were derived under initial bolus loading and constant-rate infusion plus bolus loading conditions. The concentration of enzyme-limited drug in the BAL decreased linearly with time in contrast with the concentration profile of the flow-limited drug followed by exponential functions. The perifusion model offers a quantitative understanding of the elimination kinetics of chemicals such as flow-limited and enzyme-limited drugs in a bioartificial liver and a comparison between the BAL and human liver.     

Development of a xenogeneic direct hemoperfusion method in a bioartificial liver system

 We devised a new method of xenogeneic direct hemoperfusion of a bioartificial liver support, which consists of a leukocyte adsorbent column, an immunoglobulin adsorbent column, and a substitute unit for hepatic function. Using this method in a hybrid bioartificial liver system incorporated with a nonwoven fabric bioreactor containing porcine hepatocytes, we successfully performed xenogeneic direct hemoperfusion in a canine liver failure model for 3 h without hyperacute rejection. Adequate ammonia detoxification was exhibited, and no findings of hepatocyte destruction by leukocytes or immunological proteins were detected in the canine blood analysis. Beneficial effects were also detected in a significant increase in Fischer's ratio and a decrease in intracranial pressure, indicating that our system could contribute to recovery from hepatic coma in patients with severe liver failure. Received: April 23, 2002 / Accepted: July 16, 2002 Acknowledgments We wish to thank Mr. Nobutaka Furuya from the Institute for Animal Experimentation, University of Tokyo, for his expert technical assistance in treating experimental animals. We also wish to thank Mr. Ken Shibata from Tokyo Medical Service for his excellent technical assistance in operating the perfusion equipment. We also wish to thank Dr. Wendy Gray, MD, for her excellent work in revising our English. This study is supported financially by grants in aid for scientific research (11357010, 1999–2001, and 12309003, 2000–2002) and a grant in aid for advanced forefront medical development (2000–2002). Correspondence to:K. Naruse     

The optimal hepatocyte density for a hollow-fiber bioartificial liver

A bioartificial liver (BAL) based on viable porcine hepatocytes can serve as a bridge to liver transplantation in patients with acute liver failure (ALF). To support liver functions, an adequate mass of hepatocytes is needed, which depends upon the cell density in the BAL device. This study evaluated the optimal density of hepatocytes within BAL devices that were constructed by perfusing porcine hepatocyte suspensions mixed with cytodex-3 into polysulfon hollow-fibers. The BAL devices were prepared with 6 different cell densities. The mass of hepatocytes in each device was evaluated for (a) cell viability, (b) ability to degrade diazepam, (c) ability to synthesize urea, (d) incorporation of [3H]-leucine into protein, (e) glucose-6-phosphatase activity, (f) total RNA content, and (g) p53 gene expression. Hepatocyte viability was about 90% in each device. With increasing hepatocyte density, the diazepam concentration in the medium decreased from 9.26 +/- 0.96 mg/L at 1 x 10(5) cells/ml to a minimum of 5.25 +/- 1.02 mg/L at 5 x 10(6) cells/ml and thereafter remained at low levels. Urea production and [3H]-leucine incorporation into protein increased progressively until the cell density reached 5 x 10(6)/ml and thereafter remained at high levels. Glucose-6-phosphatase activity and total RNA content stayed at high levels until the cell density reached 5 x 10(6)/ml and then progressively decreased. p53 gene expression differed from the other parameters, since it increased only when the cell density reached 5 x 10(7)/ml. In conclusion, the density of 5 x 10(6) cells/ml is a critical inflection point for most of the functional parameters, although p53 gene expression is not elevated at this cell density. These findings suggest that 5 x 10(6) cells/ml is the optimal hepatocyte density in the hollow-fiber BAL device.     

Critical issues in bioartificial liver development.

End-stage liver disease accounts for over 30,000 deaths annually in the United States. Orthotopic liver transplantation is the only clinically proven treatment for patients with end-stage liver failure. A limitation of this therapy is a shortage of donor organs available. This donor organ shortage is exacerbated by the fact that the number of patients listed for transplantation has continued to increase. As a result, there has been a continuing increase in the number of patients who die waiting for a donor liver. Extracorporeal bioartificial liver devices consisting of viable hepatocytes have the potential to provide temporary support for patients with fulminant hepatic failure, thereby serving as a "bridge" to transplantation. In some patients, this temporary support would allow the native liver to regenerate function, eliminating the need for transplantation and the resulting life-long immunosuppressive therapy, all of which translates into a cost savings to the health care system. Although the bioartificial liver device is a promising technology for the treatment of liver failure, significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. An overview of the critical issues in the development of bioartificial liver devices is discussed.     

共同培养在生物人工肝中的应用

急性肝功能衰竭(AHF)病人死亡率较高,目前主要的治疗方法是肝移植,但由于供体不足,限制了它的临床应用.生物人工肝在一定程度上能替代肝移植,维持急性肝衰竭病人的生命,因此已受到越来越多的关注.生物人工肝中的肝细胞培养有多种方法,其中把肝细胞与非实质细胞共同培养,可以使肝细胞的存活时间、分化能力及合成代谢功能等有很大提高.这一发现为生物人工肝的临床应用,提供巨大的促进作用.     

Plasma versus whole blood perfusion in a bioartificial liver assist device

The ramifications of using whole blood or plasma for perfusion off an hepatocyte containing bioartificial liver bioreactor in which the hepatocytes are separated by a membrane or other physical barrier from the perfusate stream on the rate of change of patient blood concentrations are explored through dynamic modeling of whole blood perfusion as a two compartment system (patient tissue and blood compartments), and plasma perfusion as a three compartment system (patient tissue and blood compartments, and a plasma reservoir). The whole blood perfusion model is described by three dimensionless parameters: the Damkohler number, Da, which represents the ratio of the rate of conversion by the bioreactor to the rate of perfusion; kappa, which represents the ratio of the rate of internal reequilibration between the tissue and blood compartments and the rate of perfusion; and Vtb, the tissue/blood volume ratio. The plasma perfusion model has three additional dimensionless parameters: f, the fraction of plasma withdrawn from the blood in a plasma separator; alpha, the ratio of the plasma perfusion rate in the bioreactor to the blood draw rate; and Vbr, the blood/plasma reservoir volume ratio. Within the physiologic range of parameters, the rate of reduction in blood concentration in both the whole blood-perfused and plasma-perfused systems are sensitive to Damkohler number up to Da approximately 2. Neither system is sensitive to variations in kappa, and the plasma perfusion system has little sensitivity to alpha. Given bioreactors of equivalent activity, a greater rate of blood concentration reduction and lower endpoint blood concentration at equivalent perfusion times will be achieved with whole blood perfusion. There are two physical reasons for this. The first is that the plasma perfused system is only processing a fraction, f, of the blood compared with the whole blood perfusion system. The second reason is that, although the blood-perfused system is limited by overall bioreactor performance, the plasma-perfused system is mass transfer limited to the rate of blood concentration dilution into the plasma reservoir rather than limited by the overall bioreactor performance.