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BACKGROUND: Stem cells have wide application prospects in tissue engineering, regenerative medicine, cell therapy and drug research. In recent years, the applied research, such as bioartificial liver, requires large-scale and high-quality stem cells by expansion, and final obtains hepatocytes (hepatocyte-like cells) by directional differentiation. Through this way, how to efficiently obtain the differentiated cells, with excellent consistency, powerful function and uniform expression of markers, are the key problems to be solved. OBJECTIVE: To review the detection indicators and detection methods related to the process of stem cell expansion and hepatic differentiation, summarize the indicators and methods that have been applied to online detection, discuss the limitations of some indicators and methods applied to online detection, and prospect the indicators and detection technologies expected to be applied to online detection in the future. METHODS: PubMed, Web of Science, Elsevier and CNKI databases were retrieved for the articles concerning stem cell expansion and hepatic differentiation published from 1990 to 2019. The literature related to stem cell expansion and hepatic differentiation was collected. The keywords were “stem cells; expansion; hepatic differentiation; monitoring; real-time online” in English and Chinese, respectively. A total of 93 articles were searched, and finally 53 eligible articles were selected and reviewed. RESULTS AND CONCLUSION: There are many detection indicators and detection methods for stem cell expansion and hepatic differentiation. Among them, the detection indicators of the expansion process are mainly related to the maintenance of pluripotency, metabolic activity, survival rate, and cancer transformation trend of stem cells. The process of hepatic differentiation varies with cell types, in which, the pluripotent stem cells and the mesodermal lineage adult pluripotent stem cells should differentiate into the endoderm-oriented cells and then the mature hepatocytes (hepatocyte-like cells), while the endodermal lineage adult pluripotent stem cells mainly composed of hepatic stem/progenitor cells can directly differentiate into mature hepatocytes (hepatocyte-like cells). The specific markers, survival rate and metabolic activity of cells in different stages of differentiation are the focus of detection. At present, although the detection methods based on large-scale biochemical detection and analysis equipment have high reliability, they face the problems of poor real-time performance, high cost and difficulties in integration and miniaturization. Future concerns are focused on the screening of key detection indicators, quantification of detection criteria and realization of automatic online detection during stem cell expansion and hepatic differentiation.
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Acute liver failure (ALF) is a severe clinical syndrome with rapid progression, poor prognosis and high mortality. Liver transplantation is the most effective option for the treatment of ALF, but only a few ALF patients can receive the donor liver in time due to the insufficient supply of the organ. Stem cell-related techniques, including stem cell transplantation, bioartificial liver, etc., with their roles of detoxication, synthesis, metastasis and secretion, have brought some hope for the solution of this problem. This article presents an overview of the three aspects as follows: mechanisms and clinical translation of stem cell transplantation for ALF, establishment of the coculture system for mesenchymal stem cells with porcine hepatocytes and clinical translation of the coculture-based bioartificial liver, and establishment of human-induced hepatocytes and clinical translation of bioartificial liver based on human-induced seed cells.
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Objective@#To compare the efficacy and safety of plasma exchange (PE) combined with double plasma absorption and simple PE in the treatment of acute-on-chronic liver failure.@*Methods@#We retrospectively analyzed 251 cases of acute-on-chronic liver failure treated with artificial liver treatment since January 2015. Changes in clinical manifestations, laboratory tests, and complications of the patients before and after different modes of treatment were compared and short-term efficacy was tracked. In accordance with different data, t-test, Pearson's chi-squared test and Fisher's exact test were used for statistical analysis.@*Results@#The effectiveness of low-volume PE combined with double plasma molecular adsorption system (DPMAS) and equal amount of PE combined with DPMAS was significantly better than simple PE (83.7%, 84.05% and 82.15 vs 55.6%, P < 0.05) in early stage of liver failure. In late-stage of liver failure, there was no significant difference in the treatment efficiency of each group (P > 0.05). Bilirubin and bile acid levels were significantly decreased in combined treatment groups than that to simple PE group (P < 0.05). PTA and albumin improvement rate of DPMAS PE groups were significantly lower than that of simple PE group (P < 0.05). There was no statistical difference in adverse reactions between each group.@*Conclusion@#PE combined with DPMAS improves the treatment efficiency of early hepatic failure and decrease dosage of plasma when compared with simple PE. A beforehand DPMAS treatment after PE treatment can improve the adverse effects of DPMAS on blood coagulation function and albumin levels.
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Bioartificial liver support system (BALSS) provides a new way to treat liver failure and leaves more time for patients who are waiting for liver transplantation. It has detoxification function as well as the human liver, at the same time it can provide nutrition and improve the internal environment inside human body. Bioreactors and hepatocytes with good biological activity are the cores of BALSS which determine the treatment effect. However, in the course of prolonged treatment, the function and activity of hepatocytes might be greatly changed which could influence the efficacy. Therefore, it is very important to detect the status of the hepatocytes in BALSS. This paper presents some common indicators of cell activity, detoxification and synthetic functions, and also introduces the commonly detection methods corresponding to each indicator. Finally, we summarize the application of detection methods of the hepatocyte status in BALSS and discuss its development trend.
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Objective To investigate the potential of substrate elasticity in regulating rapid differentiation of HepaRG cells into hepatocyte-like cells ,and further provide hepatocytes for bioartificial liver. Methods The substrate elasticity was divided into 4 groups. The expressions of albumin(ALB)were detected by albumin-green fluorescent protein-reporter system (ALB-GFP-reporter system) and Image J software;the cell morphology was observed by microscope and the amounts of cell were detected by cell Titer-Blue cell viability assay kit (alamar blue). Results The results of ALB showed that at the 4th hour,the expressions of ALB inside the HepaRG cells between 4s group and 8s group,16 s group and Glass group were not statistically different (t = 0.791,1.389, 2.481,P>0.05);at the 4th day,the expressions of 4s group had statistical differences in comparison with those of 16s group and Glass group(t = 12.41,12.52,P 0.05);at the 7th day,the expressions of 4s group were statistically different from those of 8s group,16s group and Glass group(t=3.266,6.725,8.005,P0.05). Conclusion Soft substrate can promote differentiation of HepaRG cells.
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The liver is the largest organ in the body; it has a complex architecture, wide range of functions and unique regenerative capacity. The growing incidence of liver diseases worldwide requires increased numbers of liver transplant and leads to an ongoing shortage of donor livers. To meet the huge demand, various alternative approaches are being investigated including, hepatic cell transplantation, artificial devices and bioprinting of the organ itself. Adult hepatocytes are the preferred cell sources, but they have limited availability, are difficult to isolate, propagate poor and undergo rapid functional deterioration in vitro. There have been efforts to overcome these drawbacks; by improving culture condition for hepatocytes, providing adequate extracellular matrix, co-culturing with extra-parenchymal cells and identifying other cell sources. Differentiation of human stem cells to hepatocytes has become a major interest in the field of stem cell research and has progressed greatly. At the same time, use of decellularized organ matrices and 3 D printing are emerging cutting-edge technologies for tissue engineering, opening up new paths for liver regenerative medicine. This review provides a compact summary of the issues, and the locations of liver support systems and tissue engineering, with an emphasis on reproducible and useful sources of hepatocytes including various candidates formed by differentiation from stem cells.
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Adult , Humans , Bioprinting , Extracellular Matrix , Hepatocytes , Incidence , Liver Diseases , Liver Transplantation , Liver , Liver, Artificial , Regenerative Medicine , Stem Cell Research , Stem Cells , Tissue Donors , Tissue EngineeringABSTRACT
Objective To design a new type of hybrid bioartificial liver (HBAL), evaluate its efficacy in vitro, and explore the feasibility in clinical application.Methods CL-1 human hepatocytes were cultured on microcarriers for 5 days, when cell count reached about 4.0 × 109 with cell density of about 4.0 × 107/ml.CL-1 cells cultured on microcarriers in home-made bioreactor constitute the biological part of the HBAL.The abiotic part included blood perfusion and bilirubin adsorption, and blood pump was employed as the circulation driver, which were parts of HBAL.The changes of the concentrations of indirect bilirubin (UBD), chenodeoxycholic acid (CDCD), cholic acid (CA), blood ammonia (AA), AST, ALT and LDH were observed under the condition of in vitro circulation.Meanwhile, the function, morphology and the cell activity of CL-1 cells were also observed.Results After in vitro circulation for 24 h, the concentrations of UBD, CDCD, CA and AA significantly decreased from (335.3 ± 6.0) μmol/L, (395.0 ± 5.6) μmol/L, (155.7 ± 4.5) μmol/L, (39.0 ± 2.6) μmol/L at 0 h to (106.0 ± 10.9) μmol/L, (131.8 ± 28.7) μmol/L, (42.2 ± 7.3) μmol/L, (3.5 ± 1.0) μmol/L, respectively.At 48 h, ALT, AST and LDH significantly increased from (25.9 ± 4.2) IU/L, (22.0 ± 3.6) IU/L, (0.28 ± 0.09) μmol/L to (31.0 ± 2.6) IU/L,(31.6 ± 8.0) IU/L, (0.41 ± 0.12) μmol/L, meanwhile the count and vitality of CL-1 cells were significant declined.Conclusions (1) In the new HBAL system, CL-1 cells can keep its viability and function in vitro;and (2) the HBAL appears to be effective in purifying the serum in liver failure simulation model by clearing out non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride, which seems to be a promising therapeutic option.
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Artificial liver support system (ALSS),as a means of bridging patients with liver failure to transplantation,has been widely used at home and abroad.However,clinical trails show that different ALSS may have different therapeutic effects.This review briefly introduces the widely-used non-biological liver support technologies and extensively-studied biological liver support technologies,and tries to give an overview on their impact on biochemical markers and survival.
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Objective To investigate the safety and therapeutic effects of the novel bioartificial liver (BAL) combined with liver transplantation in patients with liver failure.Method Twenty-two patients with liver failure were admitted.Ten of them were treated with the novel BAL 24 h before liver transplantation,while the rest 12 served as controls and received liver transplantation only.The clinical signs and symptoms,liver function,ammonia,coagulation function and complete blood count were evaluated before,during and after the treatment.Levels of xenoantibodies (IgG and IgM) were detected by ELISA kit.Titers of complement were quantified by CH50 kit.DNA in the collected PBMCs was extracted for PCR with PERV specific primers and the porcine specific primer Sus scrofa cytochrome B.The RT activity was detected as well.The operation related information was recorded,such as operation success rate,operative time,cold ischemia time,bleeding volume in operation and liver function.Result All treatment procedures were completed successfully without any adverse reaction.In the BAL group,the clinical symptoms such as acratia,anorexia and abdominal distension were improved,as well as the stage of hepatic encephalopathy and the results of experimental tests such as liver function,ammonia,and coagulation function.No PERV infection and no obvious changes of the IgG,IgM and CH50 levels were detected in patients plasma.All patients were successfully bridged to modified pig-back liver transplantation and recovered.There were no differences in operative time and cold ischemia time (P＞0.05).However,bleeding volume was different in these two groups (P＜0.05).The liver function was improved significantly in BAL group than the control group after liver transplantation (P＜0.01).Conclusion The novel BAL could improve the internal environment of patients with liver failure,and enhance the safety and efficiency of liver transplantation.The novel BAL combined with liver transplantation could be an effective therapy for patients with liver failure.
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BACKGROUND:Bioartificial liver could partial y replace the major liver functions, including detoxification, synthesis, secretion and biotransformation. OBJECTIVE:To use bibliometric indexes to track study focuses on bioartificial liver, and to investigate the relationships among geographic origin, impact factors, and highly cited articles indexed in Web of Science. METHODS:A list of citation classics for bioartificial liver was generated by searching the database of Web of Science-Expanded using the terms“artificial liver support system”or artificial liver or“bioartificial liver”. The top 33 cited research articles which were cited more than 100 times were retrieved. RESULTS AND CONCLUSIONS:Of 4 144 articles published, the 33 top-cited articles were published between 1992 and 2010. The highest citations paper was published in 2002, with a total of 668 citations, mean cited 55.67 per year. The total citations of 33 articles were 6 094 times, with a mean of 12.64 citations per article. These top-cited papers came from 11 countries, of which 12 articles came from the United States. University of Rostock led the list of classics with five papers. Harvard University and Massachusetts General Hospital ranked the second with four papers each. The 33 top-cited articles were published in 18 journals, predominantly Annals of Surgery and Hepatology, fol owed by Artificial Organs and Biotechnology and Bioengineering. Our bibliometric analysis provides a historical perspective on the progress of bioartificial liver research. Articles originating from outstanding institutions of the United States and published in high-impact journals are most likely to be cited.
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ObjectiveTo identify the factors influencing the transfer of porcine endogenous retroviruses across the membrane of a new bioartificial liver (BAL),which is a necessary caution to consider for BALs carrying porcine hepatocytes.MethodsA novel porcine BAL composed of two circuits was constructed using a plasma filter with membrane pore size of 500 nm and a plasma component separator with membrane pore sizes 10 nm,20 nm,30 nm,and 35 nm.Co-cultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors.The BAL was continuously worked for 72 hours during which the supernatant was collected from the internal and external circuits every 12 hours.PERV RNA,reverse transcriptase (RT) activity,and in vitro infectivity from the supernatant were detected.ResultsIn the plasma filter group,the PERV RNAlevels were the same in both circuits,suggesting little to no effect of the plasma filter on the PERV RNA's crossing.With plasma component separators,PERV RNA was found in the external circuits at different times without positive RT activity and HEK293 cell infection,but its level was reduced significantly.The larger the membrane pore size was,the earlier and the more RNA was detected.ConclusionsThe membrane pore size,the treatment time and the viral level in the internal circuit are contributing factors influencing the transfer of PERV RNA across the membrane in a BAL.
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Objective To investigate the influence of membrane molecular weight cut off in our bioartificial liver(BAL)system.Methods Beagle dogs were used for a model of acute liver failure through D-galactosamine administration.The acute liver failure Beagles were divided into two groups by the membrane molecular weight cut off.Group A was treated with BAL containing 200 kDa retention rating membrane.Group B was treated with BAL containing 1200 kDa retention rating membrane.Each group underwent two six-hour BAL treatments that were performed on day 1 and day 21.BAL medium were examined and levels of IgG,IgM,and complement hemolytic unit of 50％(CH50)antibodies were measured in all Beagles and.Results BAL treatment was associated with a significant decline in levels of CH50.1200 kDa group experienced a significant increase in levels of IgG and IgM after two BAL treatments.Significant levels of canine proteins were detected in BAL medium from 1200 kDa group.Conclusions Xenogeneic immune response in the BAL system was influenced by membrane molecular weight cut off.
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ObjectiveTo study the effects of membrane molecular weight cut off on a novel bioartificial liver(BAL) system.MethodsHealthy beagles underwent 6-hour treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating.The functional changes and cell viability were characterized.ResultsHepatocyte performance levels such as albumin secretion,urea synthesis and viability were significantly higher in the 200 kDa retention rating group when compared with the 1200 kDa retention rating group (P＜0.05).Significant levels of canine proteins were detected in the BAL medium from the 1200 kDa retention rating group.Fluorescence microscopy further verified that heavy deposition of canine IgG,IgM and complement (C3) on co-culture cells were obtained after BAL treatment in the 1200 kDa retention rating group.ConclusionsSmall membrane molecular weight cut off of BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improved the performance of the BAL.
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Objective To investigate cell's viability and functions when human hepatocyte cell HepG2 and microcarriers were embedded in collagen gel and cultured in vitro.Methods Ultrasound concussion crushing method was used to get nanofiber microcarrier.HepG2 and microcarriers were embedded in collagen gel and cultured in 12 d.The albumin (ALB),urea and lactate dehydrogenase (LDH) levels in the nutrient solution were measured.Results The cells in nanofiber nanofiber microcarrier/collagen gel group were gathered around the microcarrier and formatted aggregates,the ALB and urea levels increased steadily and reached maximum in day 9,then decreased; In collagen gel without nanofiber microcarrier group cells uniformly distributed,the ALB and urea levels reached maximum in day 3,then decreased rapidly,and on day 6 majority of cells dead.Conclusion Gel entrapped nanofiber microcarriers and hepatoeytes is a high-density and longtime culture method which can possibly be used in the bioartificial liver system.
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Acute liver failure is a rapidly progressive disease of the liver associated with high morbidity and mortality without liver transplantation. Although good survival after transplantation can be achieved, due to the disparity between patients awaiting transplantation and available organs, many patients die due to progression of the disease while waiting for a liver graft. To reduce the high morbidity and mortality associated with acute liver failure, attempts have been made during the last several decades to develop a temporary liver support system, such as artificial and bioartificial livers. The artificial liver is a non-biological device mainly aimed at the removal of accumulated toxins during liver failure, and the bioartificial liver is a biological device that has bioreactors containing living hepatocytes which provide both biotransformation and synthetic liver functions. There are currently 3 artificial livers available in the market that have been actively used in the clinical field, and 11 bioartificial livers that have been developed and have undergone clinical trials. In this article, we will discuss about the 3 artificial liver devices and 5 bioartificial liver systems that are the most advanced and have been widely evaluated clinically. Also, the characteristics and the preclinical data of the first bioartificial liver system developed in Korea that is currently under clinical investigation, will be discussed.
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Humans , Alginates , Bioreactors , Biotransformation , Glucuronic Acid , Hepatocytes , Hexuronic Acids , Korea , Liver , Liver Failure , Liver Failure, Acute , Liver Transplantation , Liver, Artificial , TransplantsABSTRACT
Objective As an effective means of liver function support for acute liver failure, bioartificial liver has seen great progress in recent years. However, the development of this type of device is currently hindered by limited oxygen transport to cultured hepatocytes. In this study we try to resolve this problem by supplementing the circulating medium of the bioreactor with red blood cells.Methods Freshly isolated primary porcine hepatocytes were inoculated into our newly designed bioreactor and were divided into two groups: RPMI1640 was circulated in the control group and porcine red blood cells were added into the culture medium in the experimental group. The culture media in both groups were oxygenated through extra-corporeal membrane oxygenation, and the oxygen consumption rate (OCR) in the bioreactor was measured by a blood gas analyzer. Liver-specific functions and glucose consumption were also determined. Results The OCR of the experimental group was 1.5 fold that of the control group, and the glucose consumption rate was twice that of the control group. The liver-specific functions of the experimental group were significantly higher than those of the control group in terrns of albumin secretion and urea synthesis. Conclusion Supplementing the circulating medium of the bioreactor with red blood cells can significantly improve the oxygen supply in the bioreactor, thereby enhancing the glucose consumption and liver-specific functions of hepatocytes. This method is convenient and effective, and is expected to be an effective means to resolve the problems of oxygen supply in the bioreactor.
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As a safe and effective treatment method of fulminic hepatic failure and end stage liver disease, hepatocyte transplantation has been widely verified in many animal experiment and clinical research. It has many advantages. For instances, It is safe and easy to perform. It causes little trauma and is convenient for retransplantation. The hepatocytes are less immunogenetic and the cells from one donor liver can be used for several patients, and so on. The cell source, route of transplantation, theoretical basis and clinical application of the method are reviewed in this paper.
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Objective To probe whether the preinduced adult animal hepatocytes (AHH) by phenobarbital sodium (PBS) in vivo could boost the metabolic activity of bilirubin in vitro for seeking the optimal biomaterial for the extracorporeal bioartificial liver support system (EBLSS). Methods Twelve adult male rats with 34g mean weight were randomly divided into preinduce group and control group, the rats of the preinduce group were intraperitoneal injected daily with 45mg/kg PBS for 5 days, while the control group with normal sodium. 0.5g wet weight liver was taken from every rat after 24 hours of last administer, the suspensions were made by mixing six wet weight livers in two groups and put in the cultivate board of 96 apertures, 30ul per aperture, then 50?l jaundice serum and 20?l culture fluid were added into every aperture, which were statically cultured in the incubaton of humidity 95% at 37℃, 5% CO2 for 3 hours, and then revolved, the metabolic activity of bilirubins were determined by Beckmann auto-analyzer. Results The total bilirubins and indirect bilirubins fell 60%, 71.42% respectively in the preinduced group, and 34.78%, 54.87% in the control group (P0.05, t=1.571). There were no obvious changes in direct bilirubins in two groups. Conclusions The preinduced AHH by PBS in vivo can boost the metabolic activity of bilirubin in vitro, it may be the optimal biomaterial for EBLSS.
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The key constructional components for a bioartificial liver(BAL) include the source of hepatocyte cell line, the semi-permeable membrane or bioreactor and the delivery system. The most important part of a BAL is cell line. The properties of the hepatocyte cell line will directly affect the support efficacy of a BAL. This article reviewed the recent progress in researches on hepatocyte cell line used in BAL.
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The biological artificial liver(BAL) can offer reliable artificial liver support for the patients with hepatic failure.All BAL devices contain hepatocytes as their biological component,whose specific biological characteristics contribute to the function of the BAL.During the past two decades,various cells including human hepatocytes,heterogeneous hepatocytes and liver cell lines have been used and different culture methods have been studied to optimize the activity of the biological component.However,both functionality and safety of these cells should be improved before successful use in BAL. This paper summarizes the latest progress on it.