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.

Efects of a bioartificial liver support system on a cetaminophen-induced acute liver failure canines

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Bioartificial liver support systems for acute liver failure: A systematic review and meta-analysis of the clinical and preclinical literature

BACKGROUND Acute liver failure(ALF) has a high mortality varying from 80% to 85% with rapid progress in multi-organ system failure. Bioartificial liver(BAL) support systems have the potential to provide temporary support to bridge patients with ALF to liver transplantation or spontaneous recovery. In the past decades, several BAL support systems have been conducted in clinical trials. More recently,concerns have been raised on the renovation of high-quality cell sources and configuration of BAL support systems to provide more benefits to ALF models in preclinical experiments.AIM To investigate the characteristics of studies about BAL support systems for ALF,and to evaluate their effects on mortality.METHODS Eligible clinical trials and preclinical experiments on large animals were identified on Cochrane Library, PubMed, and EMbase up to March 6, 2019. Two reviewers independently extracted the necessary information, including key BAL indicators, survival and indicating outcomes, and adverse events during treatment. Descriptive analysis was used to identify the characteristics of the included studies, and a meta-analysis including only randomized controlled trial (RCT) studies was done to calculate the overall effect of BAL on mortality among humans and large animals, respectively.RESULTS Of the 30 selected studies, 18 were clinical trials and 12 were preclinical experiments. The meta-analysis result suggested that BAL might reduce mortality in ALF in large animals, probably due to the recent improvement of BAL, including the type, cell source, cell mass, and bioreactor, but seemed ineffective for humans (BAL vs control: relative risk(95% confidence interval),0.27(0.12-0.62) for animals and 0.72(0.48-1.08) for humans)Liver and renal functions, hematologic and coagulative parameters, encephalopathy index, and neurological indicators seemed to improve after BAL, with neither meaningful adverse events nor porcine endogenous retrovirus infection.CONCLUSION BAL may reduce the mortality of ALF by bridging the gap between preclinical experiments and clinical trials. Clinical trials using improved BAL must be designed scientifically and conducted in the future to provide evidence for transformation.

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.

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.

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.

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.

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver

AIM To develop a simplified bioartificial liver(BAL) device prototype, suitable to use freshly and preserved liver Microorgans(LMOs) as biological component. METHODS The system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hema-tocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and Via Span~? solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase(LDH) release and ammonia detoxification capacity. RESULTS The concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and Via Span~? were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls(49.3% ± 8.8%, P < 0.05). LDH release was 6.0% ± 2.3% for control LMOs, and 6.2% ± 1.7% and 14.3% ± 1.1% for BG35 and Via Span~? cold preserved LMOs, respectively(n = 6, P < 0.05). CONCLUSION This prototype relied on a simple design and excellent performance. It’s a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.

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

Bioartificial liver support for fulminant hepatic failure

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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.

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.

Construction of Bioartificial Renal Tubule Assist Device In Vitro and Its Function of Transporting Sodium and Glucose

To explore a new way of constructing bioartificial renal tubule assist device （RAD） in vitro and its function of transporting sodium （Na^＋） and glucose and to evaluate the application of atomic force microscope in the RAD construction, rat renal tubular epithelial cell line NRK-52E was cultured in vitro, seeded onto the outer surfaces of hollow fibers in a bioreactor, and then cultured for two weeks to construct RAD. Bioreactor hollow fibers without NRK-52E cells were used as control. The morphologies of attached cells were observed with scanning electron microscope, and the junctions of cells and polysulfone membrane were observed with atomic force microscope. Transportation of Na＋ and glucose was measured. Oubaine and phlorizin were used to inhibit the transporting property. The results showed that NRK-52E cells and polysulfone membrane were closely linked, as observed under atomic force microscope. After exposure to oubaine and phlorizin, transporting rates of Na^＋ and glucose were decreased significantly in the RAD group as compared with that in the control group （P〈0.01）. Furthermore, when the inhibitors were removed, transportation of Na^＋ and glucose was restored. It is concluded that a new RAD was constructed successfully in vitro, and it is able to selectively transport Na^＋ and glucose.

Current status of bioartificial kidney

Current dialysis systems are not perfect considering the low efficacy and cumbersome process of actual practice. To overcome these shortcomings, bioartificial kidneys using renal tubule cells and wearable dialysis are promising ESRD treatments available in the near future. Toward clinical application, however, there are so many obstacles for commercial use of them. In this review, current status of bioartificial kidneys such as the renal tubule cell assist device (RAD) and wearable dialysis devices is presented with the comparison to show the advantages and disadvantages.

Preclinical evaluation of the safety and effectiveness of a new bioartificial cornea

Cross-linking agents are frequently used to restore corneal properties after decellularization,and it is especially important to select an appropriate method to avoid excessive cross-linking.In addition,how to promote wound healing and how to improve scar formation require further investigation.To ensure the safety and efficacy of animal-derived products,we designed bioartificial corneas(BACs)according to the criteria for Class III medical devices.Our BACs do not require cross-linking agents and increase mechanical strength via self-cross-linking of aldehyde-modified hyaluronic acid(AHA)and carboxymethyl chitosan(CMC)on the surface of decellularized porcine corneas(DPCs).The results showed that the BACs had good biocompatibility and transparency,and the modification enhanced their antibacterial and anti-inflammatory properties in vitro.Preclinical animal studies showed that the BACs can rapidly regenerate the epithelium and restore vision within a month.After 3 months,the BACs were gradually filled with epithelial,stromal,and neuronal cells,and after 6 months,their transparency and histology were almost normal.In addition,side effects such as corneal neovascularization,conjunctival hyperemia,and ciliary body hyperemia rarely occur in vivo.Therefore,these BACs show promise for clinical application for the treatment of infectious corneal ulcers and as a temporary covering for corneal perforations to achieve the more time.

A novel bioartificial pancreas fabricated via islets microencapsulation in anti-adhesive core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized in vivo

Islets transplantation is a promising treatment for type 1 diabetes mellitus. However, severe host immune rejection and poor oxygen/nutrients supply due to the lack of surrounding capillary network often lead to transplantation failure. Herein, a novel bioartificial pancreas is constructed via islets microencapsulation in core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized in vivo. Specifically, a hydrogel scaffold containing methacrylated gelatin (GelMA), methacrylated heparin (HepMA) and vascular endothelial growth factor (VEGF) is fabricated, which can delivery VEGF in a sustained style and thus induce subcutaneous angiogenesis. In addition, islets-laden core-shell microgels using methacrylated hyaluronic acid (HAMA) as microgel core and poly(ethylene glycol) diacrylate (PEGDA)/carboxybetaine methacrylate (CBMA) as shell layer are prepared, which provide a favorable microenvironment for islets and simultaneously the inhibition of host immune rejection via anti-adhesion of proteins and immunocytes. As a result of the synergistic effect between anti-adhesive core-shell microgels and prevascularized hydrogel scaffold, the bioartificial pancreas can reverse the blood glucose levels of diabetic mice from hyperglycemia to normoglycemia for at least 90 days. We believe this bioartificial pancreas and relevant fabrication method provide a new strategy to treat type 1 diabetes, and also has broad potential applications in other cell therapies.

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.