Advances in cell sources of hepatocytes for bioartificial liver

BACKGROUND: Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells. DATA SOURCES: A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the Key words such as liver failure bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed. RESULTS: Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines the incorporation of BAL with GS-HepG2 cells or alginate encapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved. CONCLUSIONS: Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functionalhepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.

Progress in bioreactors of bioartificial livers

BACKGROUND: Bioartificial liver support systems are becoming an effective therapy for hepatic failure. Bioreactors, as key devices in these systems, can provide a favorable growth and metabolic environment, mass exchange, and immunological isolation as a platform. Currently, stagnancy in bioreactor research is the main factor restricting the development of bioartificial liver support systems. DATA SOURCES: A PubMed database search of English-language literature was performed to identify relevant articles using the keywords 'bioreactor', 'bioartificial liver', 'hepatocyte', and 'liver failure'. More than 40 articles related to the bioreactors of bioartificial livers were reviewed. RESULTS: Some progress has been made in the improvement of structures, functions, and modified macromolecular materials related to bioreactors in recent years. The current data on the improvement of bioreactor configurations for bioartificial livers or on the potential of the use of certain scaffold materials in bioreactors, combined with the clinical efficacy and safety evaluation of cultured hepatocytes in vitro, indicate that the AMC (Academic Medical Center) BAL bioreactor and MELS (modular extracorporeal liver support) BAL bioreactor system can partly replace the synthetic and metabolic functions of the liver in phase I clinical studies. In addition, it has been indicated that the microfluidic PDMS (polydimethylsiloxane) bioreactor, or SlideBioreactor, and the microfabricated grooved bioreactor are appropriate for hepatocyte culture, which is also promising for bioartificial livers. Similarly, modified scaffolds can promote the adhesion, growth, and function of hepatocytes, and provide reliable materials for bioreactors. CONCLUSIONS: Bioreactors, as key devices in bioartificial livers, play an important role in the therapy for liver failure both now and in the future. Bioreactor configurations are indispensable for the development of bioartificial livers used for liver failure, just as the modified scaffold materials available for bioreactors are favorable to the construction of effective bioartificial livers.

Bioartificial liver assist devices in support of patients with liver failure

Bioartificial liver assist devices (BALs) offer anopportunity for critical care physicians and transplantsurgeons to stabilize patients prior to orthotopic livertransplantation. Such devices may also act as a bridgeto transplant, providing liver support to patientsawaiting transplant, or as support for patients post liv-ing-related donor transplant. Four BAL devices thatrely on hepatocytes cultured in hollow fiber membranecartridges (Circe Biomedical HepatAssist (r), VitagenELADTM, Gerlach BELS, and Excorp Medical BLSS)are currently in various stages of clinical evalua-tion. Comparison of the four devices shows that severalunique approaches based upon the same overall systemarchitecture are possible. Preliminary results of theExcorp Medical BLSS Phase I safety evaluation at theUniversity of Pittsburgh, after treating four patients(F, 41, acetominophen-induced, two support periods;M, 50, Wilson's disease, one support period; F, 53, a-cute alcoholic hepatitis, two support periods; F, 24,chemotherapy-induced, one support period) are pre-sented. All patients presented with hypoglycemia andtransient hypotension at the start of extracorporealperfusion. Hypoglycemia was treated by IV dextroseand the transient hypotension responded positively toIV fluid bolus. Heparin anticoagulation was used onlyin the second patient. No serious or adverse eventswere noted in the four patients. Moderate biochemicalresponse to support was noted in all patients. Morecomplete characterization of the safety of the BLSSrequires completion of the Phase I safety evaluation.

Key challenges to the development of extracorporeal bioartificial liver support systems

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 provide in 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.

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

BACKGROUND： 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.METHODS： 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.RESULTS： 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.CONCLUSION： No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system

BACKGROUND: The demand for the clinical use of hepa- tocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes. METHODS: The domestic extracorporeal circulating perfu- sion apparatus was used to isolate and harvest swine hepato- cytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and ob- served by the trypan blue exclusion test, biochemical mea- surements, phase contrast microscopy and transmission electron micrography (TEM). RESULTS: The total yield of isolated hepatocytes reached to 1.5(±0.4)×l010 per liver with a viability of 92(±5)%, and the purity of hepatocytes reached to 98% Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotrans- ferase ( AST ), lactate dehydrogenase ( LDH ), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly. CONCLUSION: This method of procuring swine hepato- cytes could get high quality cells with active metabolic function.

A reliable graded acute liver failure model in rats: treatment with internal bioartificial liver

BACKGROUND: Appropriate animal models are impor- tant for studying acute liver failure. This study was to assess a new suitable rat model for acute liver failure. METHODS: After the right influent hepatic vessels were clamped for a period of time (45, 60 or 90 minutes respec- tively), the animal model was established by removal of the clamp for restoring blood flow of the right lobes while im- mediately removal of the median, left lateral and caudate lobes. Animal survival rate was observed in the following 14 days in each group. To study the pathophysiological chan- ges of the model, some biochemical parameters in 5 con- secutive days were evaluated in the 60-minute group. Inter- nal bioartificial liver was transplanted in the peritoneal cavi- ty to test the reversibility of the model. RESULTS: The survival rate of the models decreased, as the ischemia time of the right lobes prolonged to zero in the 90-minute group, to 50% in the 60-minute group and to 100% in the 45-minute group on the fifth day after opera- tion. The levels of ammonia, alanine aminotransferase, al- kaline phosphatase, total bilirubin and prothrombin were elevated dramatically 12 to 24 hours after operation in the 60-minute group. When internal bioartificial liver was transplanted, the survival rate increased significantly in ad- dition to the levels of ammonia and total bilirubin. CONCLUSION: A period time of ischemic injury in the right lobe followed by 70% liver resection can produce a graded acute hepatic failure model in rats.

Recent advances in the application of cultured hepatocytes into bioartificial liver

INTRODUCTIONOver the past 3 decades,various experimentalliver support systems have been studied.Early artifi-cial liver support systems included hemodialysis,ex-tracorporeal liver perfusion,human cross-circulation,charcoal hemoperfusion,hepatodialysis,fresh blood,plasma exchange transfusion etc.Thesesystems were developed to remove toxic substancesfrom the blood.Clinical trials showed that thesedetoxification systems could promote the recovery

A bioartificial transgenic porcine whole liver expressing human proteins alleviates acute liver failure in pigs

Background:Preventing heterologous protein influx in patients is important when using xenogeneic bioartificial livers(BALs)to treat liver failure.The development of transgenic porcine livers synthesizing human proteins is a promising approach in this regard.Here,we evaluated the safety and efficacy of a transgenic porcine liver synthesizing human albumin(h ALB)and coagulation factor VII(h FVII)within a bioartificial system.Methods:Tibetan miniature pigs were randomly subjected to different interventions after surgeryinduced partially ischemic liver failure.Group A(n=4)was subjected to basic treatment;group B(n=4)was to standard medical treatment and wild-type porcine BAL perfusion,and group C(n=2)was to standard medical treatment and transgenic BAL perfusion.Biochemical parameters,coagulation status,survival time,and pathological changes were determined.Expressions of h ALB and h FVII were detected using immunohistochemistry and enzyme-linked immunosorbent assays.Results:The survival time in group A was 9.75±1.26 days;this was shorter than that in both perfused groups,in which all animals reached an endpoint of 12 days(P=0.006).Ammonia,bilirubin,and lactate levels were significantly decreased,whereas albumin and fibrinogen levels were increased after perfusion(all P<0.05).h ALB and h FVII were detected in transgenic BAL-perfused pig serum and ex vivo in the liver tissues.Conclusions:The humanized transgenic pig livers could synthesize and secrete h ALB and h FVII ex vivo in a whole organ-based bioartificial system,while maintaining their metabolism,detoxification,transformation,and excretion functions,which were comparable to those observed in wild-type porcine livers.Therefore,the use of transgenic bioartificial whole livers is expected to become a new approach in treating acute liver failure.

Metabolic effects of a novel bioartificial liver on serum from severe hepatitis patients:an in vitro study

Objective To establish a novel bioartificial liver (BAL) consisting of spheroids of porcine hepatocytes in a hollow-fiber bioreactor,and to perform an in vitro study on its metabolic effects on the serum from severe hepatitis B patients. Methods Hepatocytes were isolated from pup pigs and cultured as aggregate spheroids through rotation and vibration. Phase-contrast microscopy,transmission electron microscopy,and scanning electron microscopy were used for morphological detection of hepatocyte spheroids. The hepatocyte spheroids were then transferred into the shell of a polysulfone hollow-fiber bioreactor,creating a novel BAL. Diluted serum samples of severe hepatitis B patients were circulated for 3 hours each into the bioreactor,by using an extracorporeal circulatory system. Every half hour,including both before and after perfusion,serum samples were collected to assay total bilirubin (TBIL),total protein (TP),albumin (ALB),and globulin (GLB) concentrations in order to judge the metabolic effects of this novel BAL. Results Most hepatocytes had formed spheroids with high viability after 24 hours in culture. After 3 hours of perfusion,when compared with the control group, the serum concentration of TBIL in the treatment group decreased significantly ( P <0.01),but the serum concentrations of TP and ALB increased significantly ( P <0.05). Conclusions Hepatocytes can be conveniently cultured as aggregate spheroids through a rotation and vibration method. The novel BAL is efficient in removing bilirubin from the serum of severe hepatitis B patients,and in supplying the serum with ALB. Thus,the BAL might provide effective therapy for patients with severe hepatitis B.

Bioartificial liver devices:Perspectives on the state of the art

Acute liver failure remains a significant cause of morbidity and mortality.Bioartificial liver(BAL)devices have been in development for more than 20 years.Such devices aim to temporarily take over the metabolic and excretory functions of the liver until the patients’own liver has recovered or a donor liver becomes available for transplant.The important issues include the choice of cell materials and the design of the bioreactor.Ideal BAL cell materials should be of good viability and functionality,easy to access,and exclude immunoreactive and tumorigenic cell materials.Unfortunately,the current cells in use in BAL do not meet these requirements.One of the challenges in BAL development is the improvement of current materials;another key point concerning cell materials is the coculture of different cells.The bioreactor is an important component of BAL,because it determines the viability and function of the hepatocytes within it.From the perspective of bioengineering,a successful and clinically effective bioreactor should mimic the structure of the liver and provide an in vivo-like microenvironment for the growth of hepatocytes,thereby maintaining the cells’viability and function to the maximum extent.One future trend in the development of the bioreactor is to improve the oxygen supply system.Another direction for future research on bioreactors is the application of biomedical materials.In conclusion,BAL is,in principle,an important therapeutic strategy for patients with acute liver failure,and may also be a bridge to liver transplantation.It requires further research and development,however,before it can enter clinical practice.

D-galactosamine based canine acute liver failure model

Background: Appropriate preclinical evaluation of a bioartificial liver assist device (BAL) demands a large animal model, as presented here, that demon- strates many of the clinical features of acute liver failure and that is suitable for clinical qualitative and quantitative evaluation of the BAL. A lethal canine liver failure model of acute hepatic failure that re- moves many of the artifacts evidenced in prior canine models is presented. Methods: Six male hounds, 24-30 kg, under isoflu- rane anesthesia, were administered 1.5 g/kg D- galactosamine intravenously. Canine supportive care followed a well-defined management protocol that was guided by electrolyte and invasive monitoring consisting of arterial pressure, central venous pres- sure, extradural intracranial pressure (ICP), pul- monary artery pressure, and end-tidal CO_2. The animals were treated until death-equivalent, defined as inability to sustain systolic blood pressure>80 mmHg for 20 minutes despite maximal fluids and 20 μg·kg^(-1)·min^(-1) dopamine infusion. Results: The mean survival time was 43.7±4.6 hours (mean±SE). All animals showed evidence of progressive liver failure characterized by increasing liver enzymes (aspartate transaminase from 26 to 5977 IU/L; alanine transaminase from 32 to 9740 IU/L), bilirubin (0.25 to 1.30 mg/dl), ammonia (19. 8 to 85. 3 μmol/L), and coagulopathy (pro- thrombin time from 8.7 to 46 s). Increased lability and elevations in intracranial pressures were ob- served. All animals were refractory to maintenance of cerebral perfusion pressure even with only mode- rately elevated intracranial pressure. Severe neuro- logic obtundation, seen in 2 of 6 animals, was associ- ated with elevations of ICP above 50 mmHg. Post- mortem liver histology showed evidence of massive hepatic necrosis. Postmortem blood and ascites mi- crobial growth was consistent with possible transloca- tion of intestinal microbes. Conclusions: The improved lethal canine liver failure model presented here reproduces many of the clinical features of acute liver failure. The model may prove useful for qualitative and quantitative evaluation of BALs.

Effects of plasma from patients with acute on chronic liver failure on function of cytochrome P450 in immortalized human hepatocytes

BACKGROUND: The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepatocytes in the bioreactor, and potentially reduce the efficacy of bioartificial liver devices. This study was designed to investigate the effects of plasma from patients with acute on chronic liver failure (AoCLF) on immortalized human hepatocytes in terms of cytochrome P450 gene expression, drug metabolism activity and detoxification capability. METHODS: Immortalized human hepatocytes (HepLi-2 cells) were cultured in medium containing fetal calf serum or human plasma from three patients with AoCLF. The cytochrome P450 (CYP3A5, CYP2E1, CYP3A4) expression, drug metabolism activity and detoxification capability of HepLi-2 cells were assessed by RT-PCR, lidocaine clearance and ammonia elimination assay. RESULTS: After incubation in medium containing AoCLF plasma for 24 hours, the cytochrome P450 mRNA expression of HepLi-2 cells was not significantly decreased compared with control culture. Ammonia elimination and lidocaine clearance assay showed that the ability of ammonia removal and drug metabolism remained stable. CONCLUSIONS: Immortalized human hepatocytes can be exposed to AoCLF plasma for at least 24 hours with no significant reduction in the function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and can be potentially used in the development of bioartificial liver. (Hepatobiliary Pancreat Dis Int 2010; 9:611-614)

Different Effects of Plasma from Patients with Acute on Chronic Liver Failure on the Proliferation and Biotransformation Function of C3A Cells

Objective To investigate the effects of plasma from patients with acute on chronic liver failure on the proliferation and biotransformation function of C3A cells in vitro,and provide experimental data for C3A cells to be efficiently used in the bioartificial liver system.Methods C3A cells were incubated in 100%normal human plasma(NHP)and 100%abnormal plasma(AP)from patients with acute on chronic liver failure.Growth morphology of the two groups were observed under inverted microscope and scanning electron microscope.The method of methyl thiazolyl tetrazolium(MTT)was conducted for the proliferation activities of C3A cells.The cellular apoptosis rates were assessed by the flow cytometer.The biotransformation function of cells was evaluated through diazepam metabolic amount assay.The concentrations of epithelial growth factor(EGF),transforming growth factor-α(TGF-α)and interleukin-1(IL-1)were detected in plasma of the two groups.Results A:The proliferation activities of C3A cells incubated in 100%AP for 24,48,72,96 and 120 hours were significantly higher than that in 100%NHP(P<0.01).B:Observation under the inverted microscope indicated that the cells in 100%AP were growing faster than those in 100%NHP after cells attached to the plastic at24 and 48 hours.The same phenomena was observed under the scanning electronic microscope.C:The C3A cells cultured in both groups of plasma showed the same apoptosis rate at 48 hours and there was no statistical difference.D:The diazepam metabolic value of C3A cells incubated in 100%AP for 24,72 and 120 hours were lower than that in 100%NHP and were statistically different(P<0.01).E:The concentrations of TGF-α,EGF and IL-1 in AP were significantly higher than that in NHP(P<0.01).Conclusions Compared with normal human plasma,the plasma from patients with acute on chronic liver failure has more obvious effect to facilitate the proliferation of C3A cells,but also decreases partial biotransformation function of C3A cells.

Pluripotent Stem Cell-derived Strategies to Treat Acute Liver Failure: Current Status and Future Directions

Liver disease has long been a heavy health and economic burden worldwide.Once the disease is out of control and progresses to end-stage or acute organ failure,orthotopic liver transplantation(OLT)is the only therapeutic alternative,and it requires appropriate donors and aggressive administration of immunosuppressive drugs.Therefore,hepatocyte transplantation(HT)and bioartificial livers(BALs)have been proposed as effective treatments for acute liver failure(ALF)in clinics.Although human primary hepatocytes(PHs)are an ideal cell source to support these methods,the large demand and superior viability of PH is needed,which restrains its wide usage.Thus,a finding alternative to meet the quantity and quality of hepatocytes is urgent.In this context,human pluripotent stem cells(PSC),which have unlimited proliferative and differential potential,derived hepatocytes are a promising renewable cell source.Recent studies of the differentiation of PSC into hepatocytes has provided evidence that supports their clinical application.In this review,we discuss the recent status and future directions of the potential use of PSC-derived hepatocytes in treating ALF.We also discuss opportunities and challenges of how to promote such strategies in the common applications in clinical treatments.

Developing tissue engineering strategies for liver regeneration

The regenerative function of liver can be destroyed by viral infection,drug poisoning and tumorigenesis,resulting in irreversible damage.Numerous approaches in promoting liver repair intend to replace liver transplantation,which is faced with a shortage of donors.Owing to the significant advantages in cell programming and bioscaf-fold engineering,liver tissue engineering is considered to be the most promising alternative for mimicking liver microstructure,maintaining hepatic function or implanting whole liver.Cell sources gradually develop from pri-mary hepatocytes,tumor cells,stem cell-induced cells to multiple cell coculture formats,spheroids and organoids,which have realized the improvement of cell function,overcome the problem of large-scale cell expansion and avoid the risk of immune rejection.Scaffolds,biocompatible materials are applied as cell carriers,and decel-lularized scaffolds and three-dimensional bioprinting liver pipeline structures are also used to accelerate cell colonization and proliferation.We enumerate sufficient research concerning liver tissue engineering in this re-view,including single and multiple cell sources,implantable and extracorporeal scaffold materials,and so on,providing critical conclusions and future implications of tissue engineering in liver regenerative applications.

Selectivity of biopolymer membranes using HepG2 cells

Bioartificial liver(BAL)system has emerged as an alternative treatment to bridge acute liver failure to either liver transplantation or liver regeneration.One of the main reasons that the efficacy of the current BAL systems was not convincing in clinical trials is attributed to the lack of friendly interface between the membrane and the hepatocytes in liver bioreactor,the core unit of BAL system.Here,we systematically compared the biological responses of hepatosarcoma HepG2 cells seeded on eight,commercially available biocompatible membranes made of acetyl cellulose–nitrocellulose mixed cellulose(CA–NC),acetyl cellulose(CA),nylon(JN),polypropylene(PP),nitrocellulose(NC),polyvinylidene fluoride(PVDF),polycarbonate(PC)and polytetrafluoroethylene(PTFE).Physicochemical analysis and mechanical tests indicated that CA,JN and PP membranes yield high adhesivity and reasonable compressive and/or tensile features with friendly surface topography for cell seeding.Cells prefer to adhere on CA,JN,PP or PTFE membranes with high proliferation rate in spheriod-like shape.Actin,albumin and cytokeratin 18 expressions are favorable for cells on CA or PP membrane,whereas protein filtration is consistent among all the eight membranes.These results further the understandings of cell growth,morphology and spreading,as well as protein filtration on distinct membranes in designing a liver bioreactor.