Bioartificial Liver System Research Articles

Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver

The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)-BAL during a 7-day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2-fold more ammonia and produced 3-fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5-fold higher than MPHs and 6.6-fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue-like structures inside the AMC-BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading.

In vitro effect of interleukin-3 and -6 on the hepatocyte-specific metabolic capacity of adult bone marrow-derived liver stem cells

Objective. Bone marrow-derived adult liver stem cells (BALSC) represent a potential target for future cell-based therapies. For a salvage therapy in conditions such as liver failure, it is imperative to render BALSC functional in a timely manner. The aim of this study was to investigate whether interleukin (IL)-3 and -6 are capable of enhancing the liver-specific metabolic capacity of BALSC. Material and methods. Rat BALSC were cultured alone or with syngeneic hepatocytes. IL-3 and/or -6 were used to stimulate BALSC in cultures. The urea formation capacity of cultured BALSC was then determined. Results. When compared to BALSC in culture alone, urea metabolism was enhanced by 60.0% in co-cultures (p<0.0001). IL-3 and -6 increased urea formation in BALSC by 124.0% (p<0.0001). Conclusions. IL-3 and -6 enhance urea metabolism in BALSC. These cytokines may be used to prime BALSC prior to clinical application in bioartificial liver systems or cell transplantation.

Cultivation of hepatoma cell line HepG2 on nanoporous aluminum oxide membranes

Nanoporous aluminum oxide membranes were prepared by anodic oxidation of aluminum for application as novel cell culture substrates. Self-supporting as well as mechanically stabilized nanoporous membranes were produced from aluminum plates and micro-imprinted aluminum foils, respectively. Membranes of two different pore sizes (70 and 260 nm) were selected to investigate cellular interactions with such nanoporous substrates using cells of hepatoma cell line HepG2. The membranes express excellent cell-growth conditions. As shown by scanning electron microscopy investigations, the cells could easily adhere to the membranes and proliferate during a 4 day cell culture period. The cells exhibit normal morphology and were able to penetrate into pores with a diameter of 260 nm by small extensions (filopodia). On mechanically stabilized aluminum oxide membranes it was observed that the cells even adhere to the walls of the small cavities. It was demonstrated experimentally that the nanoporous aluminum oxide membranes are well suited as substrates in cell culture model systems for metabolic, pharmacological/toxicological research, tissue engineering and studies on pathogens as well as bioartificial liver systems.

Hepatocyte transplantation: State of the art

Advances in biotechnology have allowed hepatocyte transplantation as a relevant proposition to treat liver disease. This procedure may change the crescent mortality in liver transplantation waiting lists due global organ shortage. Recent clinical trials have described promising results of hepatocyte transplantation for acute, acute-on-chronic and metabolic liver disease. In this report, we discuss progresses regarding hepatocyte culture, cryopreservation systems, hepatocyte immortalization, suitable recipient site for hepatocyte engraftment, cell differentiation and fusion into hepatocytes, current clinical trials, and summarize the bioartificial liver systems. These progressions motivate expectation concerning hepatocyte transplantation as a consistent therapy for liver disease.

Bioartificial Liver: Its Pros and Cons

Both the large variety of liver functions for maintaining body homeostasis and the proven effectivity of whole liver transplantation in the therapy of acute liver failure (ALF), are important reasons to presume that cell-free liver support systems will not be able to adequately support the failing liver. Accordingly, bioartificial liver (BAL) systems have shown their efficacy in experimental ALF models in small and large animals, and have shown to be suitable and safe in phase 1 studies in humans with ALF. However, the optimal BAL system is still under development. Important issues are the source of the cellular component and the configuration of the BAL system with regard to cell attachment, mass transfer characteristics and oxygenation at site. The deficiency of all BAL systems to excrete bile effectively is another important topic for improvement. The great challenge for the future is to develop a well-functioning and safe human hepatic cell line which can replace the widely used porcine (xenogeneic) hepatocytes. Theoretically, a combination of a cell-free liver support system and a BAL system might be optimal for the treatment of ALF patients in the near future.

Functional evaluation of a new bioartificial liver system in vivo and in vivo

To evaluate the functions of a new bioartificial liver (BAL) system in vitro and in vitro. The BAL system was configured by inoculating porcine hepatocyte spheroids into the cell circuit of a hollow fiber bioreactor. In the experiments of BAL in vitro, the levels of alanine aminotransferase (ALT), total bilirubin (TB), and albumin (ALB) in the circulating hepatocyte suspension and RPMI-1640 medium were determined during 6 h of circulation in the BAL device. In the experiments of BAL in vitro, acute liver failure (ALF) model in canine was induced by an end-side portocaval shunt combined with common bile duct ligation and transaction. Blood ALT, TB and ammonia levels of ALF in canines were determined before and after BAL treatment. During 6 h of circulation in vitro, there was no significant change of ALT, whereas the TB and ALB levels gradually increased with time both in the hepatocyte suspension and in RPMI-1640 medium. In the BAL treatment group, blood ALT, TB and ammonia levels of ALF in canines decreased significantly. The new BAL system has the ability to perform liver functions and can be used to treat ALF.

Functional evaluation of a new bioartificial liver system in vivo and in vivo

Functional evaluation of a new bioartificial liver system in vivo and in vivo

Functional evaluation of a new bioartificial liver system in vivo and in vivo

Functional evaluation of a new bioartificial liver system in vivo and in vivo

Immobilization of Cryopreserved Primary Rat Hepatocytes for the Development of a Bioartificial Liver System

Primary rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing 40% (v/v) fetal bovine serum (FBS) and 10% (v/v) dimethyl sulfoxide (DMSO) in liquid N2 for 6 months. After thawing, the cells were immobilized using 2% (w/v) alginate and 0.5% (w/v) chitosan solutions. The capacities of ammonia removal and urea synthesis of the immobilized-thawed hepatocytes were similar to those of immobilized hepatocytes without cryopreservation. This result shows that immobilized hepatocytes after cryopreservation are useful for the development of a bioartificial liver system.

Comparison between bioartificial and artificial liver for the treatment of acute liver failure in pigs

To characterize and evaluate the therapeutic efficacy of bioartificial liver (BAL) as compared to that of continuous hemodiafiltration (CHDF) with plasma exchange (PE), which is the current standard therapy for fulminant hepatic failure (FHF) in Japan. Pigs with hepatic devascularization were divided into three groups: (1) a non-treatment group (NT; n = 4); (2) a BAL treatment group (BAL; n = 4), (3) a PE + CHDF treatment group using 1.5 L of normal porcine plasma with CHDF (PE + CHDF, n = 4). Our BAL system consisted of a hollow fiber module with 0.2 mum pores and 1 multiply 10(10) of microcarrier-attached hepatocytes inoculated into the extra-fiber space. Each treatment was initiated 4 h after hepatic devascularization. The pigs in the BAL and the PE + CHDF groups survived longer than those in the NT group. The elimination capacity of blood ammonia by both BAL and PE + CHDF was significantly higher than that in NT. Aromatic amino acids (AAA) were selectively eliminated by BAL, whereas both AAA and branched chain amino acids, which are beneficial for life, were eliminated by PE + CHDF. Electrolytes maintenance and acid-base balance were better in the CPE + CHDF group than that in the BAL group. Our results suggest that PE + CHDF eliminate all factors regardless of benefits, whereas BAL selectively metabolizes toxic factors such as AAA. However since PE + CHDF maintain electrolytes and acid-base balance, a combination therapy of BAL plus CPE + CHDF might be more effective for FHF.

Mild Hypothermic Preservation for Transport Purposes of the AMC Bioartificial Liver Charged with Porcine Hepatocytes

Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37 degrees C. Bioreactors in the transport group were kept at 37 degrees C during day 1, at 15 degrees C during day 2, and again at 37 degrees C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15 degrees C and 26 degrees C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. Mild hypothermic preservation at temperatures as low as 15 degrees C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.

A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems

Extracorporeal bioartificial liver devices (BAL) are perhaps among the most promising technologies for the treatment of liver failure, but significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. One key limitation is that during BAL operation, when the device is exposed to plasma from the patient, hepatocytes are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. Based on hepatic intermediary metabolism, we have utilized mathematical programming techniques to optimize the biochemical environment of hepatocyte cultures towards the desired effect of increased albumin and urea synthesis. To investigate the feasible range of optimal hepatic function, we have obtained a Pareto optimal set of solutions corresponding to liver-specific functions of urea and albumin secretion in the metabolic framework using multiobjective optimization. The importance of amino acids in the supplementation and the criticality of the metabolic pathways have been investigated using logic-based programming techniques. Since the metabolite measurements are bound to be patient specific, and hence subject to variability, uncertainty has to be integrated with system analysis to improve the prediction of hepatic function. We have used the concept of two stage stochastic programming to obtain robust solutions by considering extracellular variability. The proposed analysis represents a new systematic approach to analyze behavior of hepatocyte cultures and optimize different operating parameters for an extracorporeal device based on real-time conditions.

Enhancement of liver-specific functions of primary rat hepatocytes co-cultured with bone marrow cells on tissue culture-treated polystyrene surfaces

Primary rat hepatocytes were co-cultured with bone marrow cells (BMCs) in a tissue culture-treated culture well (TCW) or a collagen-coated culture well (CCW). Although a medium containing serum was used, the co-cultured cells formed spheroids on the TCW within 2 days, and ammonia metabolism and albumin secretion activities were well maintained for 3 weeks. The co-cultured cells formed a monolayer on the CCW within 2 days, and liver functions were maintained for 3 weeks. The ammonia metabolism activities and albumin secretion activities of co-cultured cells on the CCWs were slightly inferior to those on the TCWs. This co-culture system of hepatocytes and BMCs is an appropriate culture system for the expression and maintenance of liver-specific functions in vitro, and is expected to be applicable to bioartificial liver systems, regenerative medicine, and liver function simulators.

Crucial Roles of Mesodermal Cell Lineages in a Murine Embryonic Stem Cell–Derived In Vitro Liver Organogenesis System

Recent studies in the field of regenerative medicine have exploited the pluripotency of embryonic stem (ES) cells to generate a variety of cell lineages. However, the target has always been only a single lineage, which was isolated from other differentiated cell populations. In the present study, we selected sublines with a high capability for differentiation to contracting cardiomyocytes and also produced germ-line chimeric mice from a parent ES line. We also succeed in establishing embryoid bodies prepared from the ES cells that differentiated into not only hepatocytes but also at least two mesodermal lineages: cardiomyocytes that supported liver development and endothelial cells corresponding to sinusoids. This allowed the development of an in vitro system using murine ES cells that approximated the events of liver development in vivo. The expression of albumin was significantly higher in cardiomyocytes that had arisen in differentiated ES cells than in those that had not. Our in vitro system for liver organogenesis consists of a blood/sinusoid vascular-like network and hepatocyte layers and shows higher levels of hepatic function, such as albumin production and ammonia degradation, than hepatic cell lines and primary cultures of murine adult hepatocytes. This innovative system will lead to the development of second-generation regenerative medicine techniques using ES cells and is expected to be useful for the development of bioartificial liver systems and drug-metabolism assays.

Bioartificial liver systems: current status and future perspective

Because the liver is a multifunctional and a vital organ for survival, the management of acute liver failure requires the support of a huge number of metabolic functions performed by the organ. Many early detoxification-based artificial liver techniques failed to treat the patients owing to the inadequate support of the many essential hepatic functions. For this reason, a bioartificial liver (BAL) comprising of viable hepatocytes on a mechanical support is believed to more likely provide these essential functions than a purely mechanical device. From 1990, nine clinical studies of various BAL systems have been reported, most of which utilize a hollow fiber technology, and a much larger number of various BAL systems have been suggested to show an enhanced performance. Safety issues such as immunological reactions, zoonosis and tumorgenicity have been successfully addressed for regulatory approval, but a recent report from a large-scale, randomized, and controlled phase III trial of a leading BAL system (HepatAssist) failed to meet our expectation of efficacy in terms of the overall survival rate. In this paper, we review the current BAL systems actively studied and discuss critical issues such as the hepatocyte bioreactor configuration and the hepatocyte source. On the basis of the insights gained from previously developed BAL systems and the rapid progress in stem cell technology, the short-term and long-term future perspectives of BAL systems are suggested.

Evaluation methods and design for bioartificial liver based on perfusion model

A bioartificial liver (BAL) is a medical device entrapping living hepatocytes or immortalized cells derived from hepatocytes. Many efforts have already been made to maintain the functions of the hepatocytes in a BAL device over a long term. However, there is still some uncertainty as to their efficacy, and their limitations are unclear. Therefore, it is important to quantitatively evaluate the metabolic functions of a BAL. In previous studies onin vitro BAL devices, two test methods, an initial bolus loading and constant-rate infusion plus initial bolus loading, were theoretically carried out to obtain physiologic data on drugs. However, in the current study, the same two methods were used as a perfusion model and derived the same clearance characterized by an interrelationship between the perfusate flow rate and intrinsic clearance. The interrelationship indicated that the CL increased with an increasing perfusate flow rate and approached its maximum value,i.e. intrinsic clearance. In addition, to set up anin vivo BAL system, the toxic plateau levels in the BAL system were calculated for both series and parallel circuit models. The series model had a lower plateau level than the parellel model. The difference in the toxic plateau levels between the parallel and series models increased with an increasing number of BAL cartridges.

Evidence for Galα(1–3)Gal expression on primary porcine hepatocytes: implications for bioartificial liver systems

To bridge acute liver failure (ALF) patients to orthotopic liver transplantation, several bioartificial liver (BAL) systems have been developed. The bio-component of most BAL systems consists mainly of porcine hepatocytes. Plasma or blood of ALF patients is perfused through the BAL thereby contacting porcine hepatocytes. Xenogeneic BAL systems may suffer from hyperacute rejection similar to whole-organ xenotransplants. Hyperacute rejection is mediated by antibodies directed against Galalpha(1-3)Gal, a carbohydrate structure present on most mammalian cells. Galalpha(1-3)Gal is produced by the enzyme alpha1,3-galactosyltansferase (alphaGal-T). Conflicting data have been published concerning Galalpha(1-3)Gal expression on hepatocytes in intact porcine liver. We investigated whether isolated porcine hepatocytes express Galalpha(1-3)Gal. Immunofluorescence, flow cytometry, RT-PCR and enzyme activity assays were performed on freshly isolated and cultured porcine hepatocytes and liver biopsies. Anti-Galalpha(1-3)Gal antibodies were measured in plasma from patients treated with BAL by ELISA. Isolated porcine hepatocytes express (alphaGal-T) at low levels and Galalpha(1-3)Gal is present in low quantities on these cells, in contrast to hepatocytes in situ. Furthermore, IgG and IgM anti-Galalpha(1-3)Gal are depleted from the plasma of ALF patients during BAL treatment. Isolation and culture of porcine hepatocytes induce Galalpha(1-3)Gal expression, which may elicit immunological responses potentially compromising BAL functionality.

An Experimental Animal Model of Fulminant Hepatic Failure in Pigs

The objective of this study was to develop an experimental animal model of fulminant hepatic failure to test the efficacy of the bioartificial liver system. The portal vein and the hepatic artery were clamped intermittently and then the hepatic artery was ligated (ligation group, n=5). Pigs whose hepatic arteries were not ligated after clamping were assigned to the non-ligation group (n=5). The biochemical changes in blood, histologic alterations of the liver and neurologic examination for pigs were checked up. All animals died within 17 hr in the ligation group. On the other hand, all animals survived more than 7 days in the non-ligation group. In the ligation group, the levels of ammonia, lactic acid and creatinine showed a progressively increasing pattern. Prothrombin time was also prolonged gradually. Cytoplasmic condensation and nuclear pyknosis of hepatocytes were detected histologically at autopsy. Neurologic findings such as decreased pain sensation, tachypnea and no light reflex of pupils were observed. The findings shown in the ligation group are similar to the clinical features of fulminant hepatic failure in human and this animal model is reproducible. Therefore, this can be a suitable animal model to evaluate the efficacy of the bioartificial liver system for treating fulminant hepatic failure.

Interleukin-3 induces hepatocyte-specific metabolic activity in bone marrow–derived liver stem cells

Bone marrow-derived adult liver stem cells (BALSC) are a promising target for the development of future cell-based therapies for a variety of liver disorders. However, the ability of stem cells to fully function, as hepatocytes, is limited and differentiation is time dependent. Therefore, it will be conducive to find a growth factor that is able to enhance liver-specific metabolic activity in freshly isolated liver stem cells. Recently, a subpopulation of BALSC was isolated and characterized (beta2-microglobulin-negative/ Thy-1-positive cells). We hypothesized that using interleukin-3 (IL-3), a hematopoietic differentiation growth factor, we may be able to enhance liver-specific metabolic activity in freshly isolated BALSC. Rat BALSC from normal and injured livers (bile duct ligated) were isolated and stimulated with IL-3 in culture. Cells were co-cultured with or without hepatocytes, separated by a semipermeable membrane. We measured the effect of IL-3 on BALSC to metabolize ammonia into urea (a liver-specific metabolic activity). IL-3 increased the ability of BALSC, purified from normal animals, to metabolize ammonia into urea by several folds. Interestingly, no such effect was found in cell cultures from bile duct-ligated animals. Additionally, co-cultures of BALSC with hepatocytes induced higher rate of ammonia metabolism, which was further enhanced by IL-3. Our study indicates that IL-3 may be used as an agent to enhance differentiation of BALSC, both qualitatively and quantitatively. It is conceivable that stem cells may undergo IL-3 priming before their clinical application in cell transplantation or bioartificial liver systems.