Hybrid Bioartificial Liver Research Articles

In vitro safety and efficacy evaluation of a novel hybrid bioartificial liver system with simulated liver failure serum.

Acute liver failure (ALF), which can potentially be treated with an artificial liver, is a fatal condition. The purpose of this study was to evaluate the safety and effectiveness of a novel hybrid bioartificial liver system (NHBLS) using simulated liver failure serum in vitro. The bioreactor in experimental group was cultivated with primary porcine hepatocytes, whereas in control group was not. Next, the simulated liver failure serum was treated using the NHBLS for 10 h. Changes in albumin (ALB), total bilirubin (TBIL), ammonia (Amm), total bile acid (TBA), creatinine (Cr), and blood urea nitrogen (BUN) were measured before treatment (0 h) and every 2 h during treatment. In addition, changes in NHBLS pressures, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lidocaine metabolism were also recorded. The NHBLS worked steadily without unexpected occurrences during the treatment. Blood culture showed no bacterial growth after 7 days, and the endotoxin level was less than 0.5 EU. The TBIL, TBA, Cr, and BUN levels in both groups were markedly lower than those at 0 h (p < 0.05). The Amm level in experimental group was significantly lower than that in control group (p < 0.05). NHBLS pressures were also stable, and the hepatocytes in the bioreactor functioned well. The preparation method for the simulated liver failure serum was optimized successfully, and the safety and effectiveness of the NHBLS in vitro were verified. Furthermore, the NHBLS significantly reduced the levels of Amm which can lead to hepatic encephalopathy.

Cell-based clinical and experimental methods for assisting the function of impaired livers – Present and future of liver support systems

Currently, one of the most serious public health issues is the increasing number of cases of chronic liver disease and cirrhosis both of which can lead to liver failure. The only effective method of treatment for this life-threatening condition remains liver transplantation. Unfortunately, the chronic shortage of transplantable organs seriously limits its accessibility to patients. Thus, tremendous research has been done to develop methods capable of replacing liver transplantation by artificial means or to create techniques to partially or fully replace liver function in patients with impaired livers, until liver regeneration or transplantation. This review article is focused on research results that utilize living cells in order to establish bridging therapies in cases of liver failure. This includes both experimental and clinically tested techniques, such as hepatocyte transplantation and usage of the hybrid bioartificial liver devices. The article also discusses research which presents the long-term culture of hepatocytes in conditions that preserve their differentiated state, which is important for such applications as drug development and toxicity testing. Last but not least, the article describes the groundbreaking efforts toward building sophisticated scaffolds for hepatocyte culture that mimic their natural environment, which are based on decellularized tissues and on three-dimensional bioprinting.

Effects of genetically modified human skin fibroblasts, stably overexpressing hepatocyte growth factor, on hepatic functions of cocultured C3A cells.

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver-specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell-cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model-C3A cells.

Current status and perspectives of artificial liver for treatment of acute-on-chronic liver failure

This article briefly introduces the mechanisms and characteristics of several new artificial liver therapies, the advantages and disadvantages of different sites for placement of the drainage tube, and commonly used anticoagulation methods, as well as the prevention and treatment of common complications of the artificial liver support system. The future direction of development of artificial liver includes new regimens for "individualized treatment" based on patients' conditions, combination of non-biological artificial liver and biological active ingredients, and development of efficient and perfected hybrid bioartificial liver, which can further improve the therapeutic outcome of liver failure.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Evaluation of a novel hybrid bioartificial liver based on multi-layer fiat-plate bioreactor

Objective To evaluate the efficacy of a novel hybrid bioartificial liver based on multilayer flat-plate bioreactor in treatment of acute liver failure.Methods The animals were divided into the hybrid bioartificial liver (HBAL) treatment group (n =8 ),the bioartificial liver (BAL) treatment group (n =8),the non-bioartificial liver (NBAL) treatment group (n =8),and the control group (n =8).Biochemical parameters and survival time were determinedt.The levels of xenoantibodies ( IgG,IgM) were determined by using enzyme linked immunosorbent assay ( ELISA ).The PERV mRNA expression in the plasma was detected by using reverse transcription-polymerase chain reaction (RT-PCR).Results Biochemical parameters were significantly decreased in all treatment groups. Analysis of survival curves showed that the 7-day survival rate was 37.5％ (3/8) in the control group,50.0％ (4/8) in the NBAL group,62.5％ (5/8) in the BAL group and 87.5％ (7/8) in the HBAL group respectively.Survival time was significantly prolonged by HBAL treatment ( P ＜ 0.05 ).Conclusion The hybrid bioartificial liver showed a great efficiency in the treatment of acute liver failure. Key words: Bioartificial liver; Acute liver failure

Evaluation of a novel hybrid bioartificial liver based on a multi-layer flat-plate bioreactor

To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure. Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected. Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 μmol/L vs 24.2 ± 6.45 μmol/L, 12.47 ± 3.62 μmol/L, 3.77 ± 1.83 μmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative. The HBAL showed great efficiency and safety in the treatment of acute liver failure.

Clinical study on hybrid bioartificial liver supporting system for acute on chronic liver failure patients

To construct an hybrid bioartificial liver supporting system, and observe its effectiveness and safety on patients with acute on chronic liver failure. Hybrid bioartificial liver supporting system (HBALSS) was constructed using bioreactor with HepG2 cells transfected with human augmenter of liver regeneration (hALR) gene. 12 acute on chronic liver failure patients were divided into 2 groups randomly. The treatment group was treated with the hybrid bioartificial liver support system. The group underwent plasma exchange was used as control. In the treatment group, four patients recovered, one patient died of hepatic encephalopathy, one patient died of hepatorenal syndrome, one patient recovered, but died of gastrointestnal bleeding after 1 year. In control group, two patients recovered, one patient underwent orthotropic liver transplantation, and three patients died of liver failure. The hybrid bioartificial liver supporting system with HepG2 cell line was established successfully and have certain safety and effectiveness on acute on chronic liver failure patients.

Porous and single-skinned polyethersulfone membranes support the growth of HepG2 cells: A potential biomaterial for bioartificial liver systems

In this study, we evaluated a porous and single-layer skin polyethersulfone (PES) membrane as a material for use in hybrid bioartificial liver support systems. The PES membrane has been characterized as a single-layer skin structure, with a rough porous surface. Specifically, we studied the ability of the human hepatoblastoma cell lines (HepG2) to adhere, grow, and spread on the PES membrane. Furthermore, we examined albumin secretion, low-density lipoprotein uptake, and CYP450 activity of HepG2 cells that grew on the membrane. HepG2 cells readily adhered onto the outer surfaces of PES membranes. Over time, HepG2 cells proliferated actively, and confluent monolayer of cells covered the available surface area of the membrane, eventually forming cell clusters and three-dimensional aggregates. Furthermore, HepG2 cells grown on PES membranes maintained highly specific functions, including uptake capability, biosynthesis and biotransformation. These results indicate that PES membranes are potential substrates for the growth of human liver cells and may be useful in the construction of hollow fiber bioreactors. Porous and single-layer skin PES membranes and HepG2 cells may be potential biomaterials for the development of biohybrid liver devices.

Construction and experimental study on off-line hybrid bioartificial liver supporting system with human liver cell line

To construct an off-line hybrid bioartificial liver supporting system with human liver cell line, and study it's effect on the plasma from patients with liver failure. We established the bioreactor using Psu-2s (Fresenius) cultured with Hep G2 cell transfected with human augmenter of liver regeneration (hALR) gene, then constructed a hybrid bioartificial liver supporting system, at last using the bioartificial liver support system to purify the plasma treated 2 hours with serum bilirubin absorbent, separated from acute on chronic liver failure patients infected by hepatitis B virus. Bioreactor was successful constructed. The cell viability in perigastrum of bioreactor is 85.2% and cell propagated rapidly. Before and after treating with bilirubin absorbent, serum total bilirubin was (176.19 +/- 54.14) micromol/L and (50.1 +/- 16.85) micromol/L respectively (P = 0.0002). While there were no significance difference in the level of albumin, urea and glucose. At the begin and end of treatment with bioartificial liver, serum total bilirubin was (50.10 +/- 16.85) micromol/L and (30.27 +/- 15.02) micromol/L respectively (P = 0.000), the urea and albumin increased, urea has significantly difference, but the change of albumin hasn't. The off-line hybrid bioartificial liver supporting system with human liver cell line were builded successfully and have synthesis and metabolism functions for acute on chronic liver failure patients.

Radial Flow Type Bioreactor for Bioartificial Liver Assist System Using PTFE Non‐Woven Fabric Coated with Poly‐amino Acid Urethane Copolymer

AbstractRecently, various types of hybrid bioartificial liver using the porcine hepatocytes have been widely studied but their clinical usage is limited by various difficulties. One of the most efficient bioreactor is the radial flow system equipped with silicon oxygenator. Here, our bioreactor system provides the oxygenator and the radial flow of medium that supplies the nutrient efficiently. The adhesion and growth of the cultured hepatocytes are greatly improved by using polytetrafluoroethylene (PTFE) non‐woven fabrics coated with poly‐amino acid urethane copolymer (PAU). Its functional activities were established after being kept for one week at the high value in this bioreactor system. These results indicate that, PTFE non‐woven fabric and radial flow technique is proven to be the best bioreactor for a hybrid artificial liver support system (HALS).

Application of hybrid bioartificial liver system using human hepatocytes cultured on microcarriers

目的 探讨微载体培养L-02人肝细胞株应用于混合型生物人工肝系统(HBLS)及其治疗实验效果.方法采用微载体Cytodex 3培养人肝细胞,经简易体外灌流实验后装配成HBLS,用于治疗急性肝衰犬;观察模型犬的临床体征、生存时间、生化指标以及死亡后各脏器的病理检查.结果 HBLS治疗组和对照组的平均生存时间分别为(7.9±2.3) h和(3.4±1.4) h,差异具有显著性(P<0.05);空舱对照组生化指标呈现进行性恶化趋势,而HBLS组治疗过程中,FBI上升,TBIL、ALT、AST、LDH、PT指标呈下降趋势;肝组织的病理显示治疗组较对照组肝细胞变性坏死程度减轻.结论微载体培养L-02人肝细胞株成功应用于HBLS,能改善急性肝衰犬的状态,延长生命,具有一定的安全性和有效性。

The bioartificial liver: state-of-the-art.

The rationale for artificial liver support is based on the hypothesis that if essential liver functions can be restored during the critical phase of liver failure, it should be possible to improve the survival of patients with severe liver disease. In the case of bridge-to-transplantation, it should provide the patient sufficient metabolic support until a donor liver can be found and transplanted. Since the management of acute liver failure requires the replacement of the liver's myriad metabolic functions, the idea of a hybrid bioartificial liver (BAL) support system has been proposed. BAL systems incorporate a biological (hepatocytes) and a synthetic housing component (plastic housing shell and semipermeable membrane) coupled in such a way as to facilitate the delivery of essential liver functions. Of the several BAL designs that have been proposed, only the capillary hollow-fiber based systems have been rapidly developed for clinical trials. Capillary hollow-fiber based BAL devices are basically off-the-shelf artificial kidney membranes that have been modified for use as an artificial liver. However, most capillary hollow-fiber based BAL designs have inherent physical limitations of total diffusion surface area and capacity for hepatocyte mass. We have proposed a novel BAL design using microencapsulated hepatocytes to overcome these physical limitations. This new BAL design (UCLA-BAL) involves the direct hemoperfusion of a packed-bed column of microencapsulated porcine hepatocytes within an extracorporeal chamber. In extensive animal studies using a well-characterized animal model fulminant hepatic failure (FHF), we demonstrated that the UCLA-BAL system had superior diffusion surface area and a higher capacity for hepatocytes compared to conventional capillary hollow-fiber based BAL devices. UCLA-BAL treatment significantly (P<0.001), improved the survival rate of FHF animals and significantly (P<0.01) prolonged the survival time of similar animals with very severe liver injury. BAL treatment was convenient, easy to operate and well tolerated, and did not adversely affect the animal's hemodynamics during treatment. We therefore suggest that the UCLA-BAL is a significant improvement over conventional, first-generation, capillary hollow-fiber BAL systems.

Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells

Background/Aims : Hybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs). Methods : Colonies of SHs were collected and replated on a collagen sponge. Hepatic functions were examined by ELISA, immunoblotting, and Northern blotting. Cells in the sponge were characterized by immunocytochemistry and transmission electron microscopy. Urea synthesis was measured and metabolization of fluorescein diacetate was examined. Results : SHs could proliferate and expand to form a hepatic organoid in the sponge. Albumin secretion and other hepatic protein production of the cells in the sponge increased with time in culture and the amounts were much larger than for those obtained from cells grown on dishes. Morphologically and functionally differentiated hepatocytes were observed and some CK19-positive cells formed duct-like structures within the sponge. Excretion of fluorescein was observed in bile canaliculi. Conclusions : Hepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs.

INVESTIGATION OF ULTIMATE STYLE FOR BIOARTIFICIAL LIVER SUPPORT

In the development of bioartificial liver support, superior efficacy, safety, and easy-operation are required. In these viewpoints, we compared several kinds of perfusion method and hepatic function unit, and determened the most desirable combination. We have established a method of xenogeneic direct hemoperfusion of bioartificial liver support, consisting of a leukocyte adsorbent column, an immunoglobulin adsorbent column, and a hepatic function unit1. We applied this method for hybrid bioartificial liver system containing a bioreactor filled with porcine hepatocytes immobilized on nonwoven polyester fabric (NWF bioreactor), and then an extrcorporeal whole swine liver system2 in experiments of canine liver failure model. In these experiments, fluent perfusion without hyperacute rejection, and adequate ammonia detoxification was exhibited. In the further step, we utilized cross plasma perfusion (CPP) method for the NWF bioreactor, whole swine liver, and hepatocyte perfusion systems3. The whole swine liver system revealed best efficacy, but showed difficulty of maintenance. The hepatocyte perfusion systems can be prepared quickly and easily, but the efficacy is inferior to others because of quantity limit of hepatocyte. We concluded that the best functional performance is given by whole swine liver system, but considering the balance of efficacy, safety, and easy-operation, the hybrid system in CPP method is most desirable. 1. International Patent Application for PCT: No. PCT/JPO1/02233. 2000.3.22 2. Domestic Patent Application: 2001–401200. 2001.12.28 3. Domestic Patent Application: 2002–236760. 2002.8.15

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.

Efficacy of Nonwoven Fabric Bioreactor Immobilized with Porcine Hepatocytes for Ex Vivo Xenogeneic Perfusion Treatment of Liver Failure in Dogs

We have developed a new bioartificial liver bioreactor filled with porcine hepatocytes immobilized on polyester nonwoven fabric (NWF). In this study, we investigated the efficacy of our hybrid bioartificial liver system incorporating the NWF bioreactors and an immunoglobulin adsorbent column for perfusion treatment in a canine liver failure model. Xenogeneic perfusion treatment for operative canine liver failure models were performed for 3 h, and survival time, intracranial pressure, and blood and cerebrospinal fluid data were documented. Treatment was carried out without obstruction by immunological rejection when immunoglobulin adsorbent columns were used with the NWF bioreactors in combination. Dogs treated with this system exhibited a restricted increase of intracranial pressure and significant compensatory effects on blood and cerebrospinal amino acid imbalances as shown by a significant improvement of Fischer's ratio. On the other hand, relatively low capacity for ammonia elimination was shown as compared with homologous direct hemoperfusion.

BIOARTIFICIAL LIVER DEVICES

A current treatment for acute liver failure, the hybrid bioartificial liver (BAL) device, includes biologically active cells in mechanical systems. A number of BAL devices are in development to serve as a bridge to transplant for patients with acute liver failure. Most of these devices, including ours, employ cultured cells in hollow fiber bioreactors. The considerations that attend the development of BAL devices will be discussed in this presentation. These considerations include cellular component, membrane component, and BAL system configuration. BAL devices based on hollow fiber bioreactors currently show the most promise, and available results to date will be reviewed. A majority of the BAL devices currently undergoing clinical trials utilize xenogeneic, porcine hepatocytes. The risks of using xenogeneic treatments have yet to be defined. Therefore, a stringent manufacturing process is required to ensure consistent quality and safety. Furthermore, the potential risk of cross-species transmission of possibly infectious, adventitious agents, such as porcine endogenous retrovirus, has raised safety concerns. The current status of xenogeneic BAL technology and regulations will be addressed in addition to public safety concerns and bioethical issues associated with BAL treatment. Finally, the technological experience gained from past and present BAL systems will be summarized and the future of BAL technology discussed with reference to performance and public safety.

Development and characterization of a hybrid bioartificial liver using primary hepatocytes entrapped in a basement membrane matrix.

For the development of a bioartificial liver (BAL) support device, it is most important to establish highly differentiated liver cells cultured at high density. When rat hepatocytes were cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, their rates of albumin secretion were very high, as measured by ELISA, and these high rates were maintained for more than three weeks of culturing. This level of activity greatly exceeded that of hepatocytes cultured on a plastic substratum, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), on a single layer of collagen, or in a collagen sandwich culture. In an in vitro perfusion experiment, rat hepatocytes rapidly and completely removed ammonia from Eagle's MEM supplemented with 0.2 mM NH4Cl, although ammonia levels of the medium serially increased in modules containing HepG2 cells. A hybrid liver support system was developed and consisted of plasma perfusion through porous hollow fiber modules inoculated with 10 billion porcine hepatocytes entrapped in EHS gel. This system was applied to pigs with ischemic liver failure 8 hr after creation of a portocaval shunt and hepatic devascularization. In animals treated with the BAL support system, blood bicarbonate levels were increased immediately after treatment, and hemodynamic stability was improved. In control pigs, on the other hand, blood bicarbonate levels and blood pressure remained low. Plasma levels of ammonia and lactate decreased in pigs treated with the BAL device, but not in control animals. These results indicate that primary hepatocytes outperform HepG2 cells as a source of biotransformation functions in a BAL system and that the use of a BAL support device in combination with a hollow fiber module and hepatocytes entrapped in EHS gel has potential advantages for clinical use in patients with fulminant hepatic failure.

Extracorporeal tissue engineered liver-assist devices.

The treatment of acute liver failure has evolved to the current concept of hybrid bioartificial liver (BAL) support, because wholly artificial systems have not proved efficacious. BAL devices are still in their infancy. The properties that these devices must possess are unclear because of our lack of understanding of the pathophysiology of liver failure. The considerations that attend the development of BAL devices are herein reviewed. These considerations include choice of cellular component, choice of membrane component, and choice of BAL system configuration. Mass transfer efficiency plays a role in the design of BAL devices, but the complexity of the systems renders detailed mass transfer analysis difficult. BAL devices based on hollow-fiber bioreactors currently show the most promise, and available results are reviewed herein. BAL treatment is designed to support patients with acute liver failure until an organ becomes available for transplantation. The results obtained to date, in this relatively young field, point to a bright future. The risks of using xenogeneic treatments have yet to be defined. Finally, the experience gained from the past and current BAL systems can be used as a basis for improvement of future BAL technology.