Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Cell-based therapy of acute liver failure: The extracorporeal bioartificial liver

The need for an alternative ttreatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporalily providing liver functions are being actively tested. Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function. An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation. Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines. Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We have isolated normal porcine hepatocytes by collagenase perfusion of the liver. Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor. Porcine hepatocytes expreessed liver-specific functions at high levels, particularly protein neosysnthesis and enzymatic activities involved in detoxication and biotransformation processes. In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins. This system represents a promising tool for the design of anoartificial liver in human beings.Abbreviations ALF acute liver failure - EBAL extracorporeal bioartificial liver - OLT orthotopic liver transplantation     

Perfusion of 3D encapsulated hepatocytes—A synergistic effect enhancing long‐term functionality in bioreactors

Long‐term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day⁻¹, for three operative weeks. Perfusion feeding led to a 10‐fold improvement in albumin synthesis in bioreactors containing non‐encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10⁻⁹ cm² s⁻¹ and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver‐specific functions of long‐term hepatocyte spheroid cultures. Biotechnol. Bioeng. 2011; 108:41–49. © 2010 Wiley Periodicals, Inc.     

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.     

In vitro assessment of encapsulated C3A hepatocytes functions in a fluidized bed bioreactor

In the present in vitro model, the authors intended to assess viability and functionality of hepatocytes encapsulated into alginate beads and submitted to a fluidized bed motion in a bioreactor. Human immortalized C3A line was chosen as cell model. Two controls consisting of (1) cells cultured on flasks and (2) cells encapsulated in alginate beads under static conditions were implemented. The cell functions studied were total protein, albumin, urea, and ammonia synthesis, as well as ammonia removal in the case of overdose. The comparison among the three cases studied showed that the three-dimensional structure of alginate offered a suitable environment for cell functions. In addition, the fluidized bed bioreactor enhanced the mass transfer and thus increased the amount of species released out of the beads, as compared with the static case. Ammonia detoxification only appeared reduced by encapsulation. The concept of a fluidized bed bioartificial liver was thus validated by this in vitro model, which indicated that cell functions could be efficiently retained. In addition, as far as urea and protein synthesis and release were concerned, the use of the C3A cell line, in combination with encapsulation and fluidization technology, offered a real potentiality for the purpose of extracorporeal liver supply.     

Evaluation of a hepatocyte-entrapment hollow fiber bioreactor: a potential bioartificial liver

We have developed a hepatocyte entrapment hollow fiber bioreactor for potential use as a bioartificial liver. Hepatocytes were entrapped in collagen gel inside the lumen of the hollow fibers. Medium was perfused through the intraluminal region after contraction of the hepatocyte-entrapment gel. Another medium stream, comparable to the patient's blood during clinical application, passed through the extracapillary space. Viability of hepatocytes remained high after 5 days as judged by the rate of oxygen uptake and viability staining. Urea and albumin synthetic activities were also sustained. Transmission electron microscopic examination demonstrated normal ultrastructural integrity of hepatocytes in such a bioreactor. With its sort-term, extracorporeal support of acute liver failure, the current bioreactor warrants further investigation. (c) 1993 John Wiley & Sons, Inc.     

Circulating monocytes accelerate acute liver failure by IL‐6 secretion in monkey

Acute liver failure (ALF) is associated with high mortality, and a poor understanding of the underlying pathophysiology has resulted in a lack of effective treatments so far. Here, using an amatoxin‐induced rhesus monkey model of ALF, we panoramically revealed the cellular and molecular events that lead to the development of ALF. The challenged monkeys with toxins underwent a typical course of ALF including severe hepatic injury, systemic inflammation and eventual death. Adaptive immune was not noticeably disturbed throughout the progress of ALF. A systematic examination of serum factors and cytokines revealed that IL‐6 increase was the most rapid and drastic. Interestingly, we found that IL‐6 was mainly produced by circulating monocytes. Furthermore, ablation of monocyte‐derived IL‐6 in mice decreased liver injury and systemic inflammation following chemical injection. Our findings reveal a critical role of circulating monocytes in initiating and accelerating ALF, indicating a potential therapeutic target in clinical treatment for ALF.     

Microfabricated grooved substrates as platforms for bioartificial liver reactors

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.     

Perfusion flow rate substantially contributes to the performance of the HepaRG‐AMC‐bioartificial liver

Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver‐failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC‐BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β‐hydroxylation of testosterone showed 2–20‐fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG‐BAL); and (3) in the current set‐up the mass balance of substrate, metabolite, or cell damage markers between in‐and out‐flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG‐BAL). Biotechnol. Bioeng. 2012; 109: 3182–3188. © 2012 Wiley Periodicals, Inc.     

Improvement of serum amino acid profile in hepatic failure with the bioartificial liver using multicellular hepatocyte spheroids

We designed a bioartificial liver support system in which encapsulated multicellular spheroids of rat hepatocytes were utilized as a bioreactor in a hollow fiber cartridge. The spheroids, formed in a positively charged polystyrene dish that contained hormonally defined medium, were encapsulated into microdroplets of agarose that contained about 9 x 10(7) rat hepatocytes. The medium, including 150 mL reservoir volume, was circulated in a closed circuit in which the cartridge was inserted. The pH and levels of dissolved oxygen were monitored and automatically regulated so that they were maintained within a constant range for 72 h. Albumin accumulated in the circuit at the rate of 2.0 mg/L/h in this system. When the bioreactor cells in the system were replaced with Hep G2 cells, a human hepatoblastoma cell line, albumin accumulated at the rate of 0.15 mg/L/h. The spheroids of primary culture hepatocytes had 13 times higher albumin-producing capacity than the aggregates of Hep G2. The serum of a patient with fulminant hepatic failure was circulated in this system with the spheroids of primary culture hepatocytes. The concentration of branched amino acid (BCAA) in the circuit significantly increased during the 48 h circulation, while the concentration of aromatic amino acid (AAA) and methionine decreased. The ratio of BCAA/AAA increased from 0.640 to 0.772, indicating that the hepatocyte spheroids had improved the imbalance of the amino acid profile in the serum. These findings indicate that this system may be a useful model for an artificial liver support. (c) 1996 John Wiley & Sons, Inc.     

Gas‐permeable membranes and co‐culture with fibroblasts enable high‐density hepatocyte culture as multilayered liver tissues

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

High level benzodiazepine and ammonia clearance by flat membrane bioreactors with porcine liver cells

The onset of hepatic encephalopathy is a multifactorial process in which endogenous benzodiazepines and hyperammonemia play a pivotal role. The treatment of comatose states in liver failure is one of the major functions of a bioartificial liver. A controlled study demonstrating the capacity of a large scale bioartificial liver to detoxify benzodiazepines could be a crucial prerequisite to break this circle of events leading to coma. The aim of this study was therefore to expose the bioreactor to high levels of benzodiazepines and ammonia for evaluation of its detoxifying capacity. We have developed a novel and unique device reconstructing the plate architecture of the liver. Porcine hepatocytes were co-cultured with non-parenchymal cells. We investigated benzodiazepine metabolism using diazepam as model drug. The bioreactor was also loaded with high levels of ammonia and ammonia clearance as well as urea secretion with ammonia challenge were investigated. Albumin secretion was analysed in parallel as a control viability and tissue specific secretory parameter. The results clearly show that the velocity of diazepam turnover increases between day 1 and 2 and stabilises at high levels. Typical diazepam metabolites including temazepam, N-desmethyl-diazepam and oxazepam were generated. Cell specific functions, including albumin secretion, were comparable to an in vivo liver. We conclude that the flat membrane bioreactor used as bioartificial liver has the potential to detoxify diazepam and ammonia at significant amounts. Maintenance of monoxygenase activities in vitro is one of the strongholds of the bioreactor concept presented in this study.     

Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver

A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure. The device is an automated mammalian cell culture system supporting 6-7 × 109 porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.     

Application of mesenchymal stem cell exosomes and their drug‐loading systems in acute liver failure

Stem cell exosomes are nanoscale membrane vesicles released from stem cells of various origins that can regulate signal transduction pathways between liver cells, and their functions in intercellular communication have been recognized. Due to their natural substance transport properties and excellent biocompatibility, exosomes can also be used as drug carriers to release a variety of substances, which has great prospects in the treatment of critical and incurable diseases. Different types of stem cell exosomes have been used to study liver diseases. Due to current difficulties in the treatment of acute liver failure (ALF), this review will outline the potential of stem cell exosomes for ALF treatment. Specifically, we reviewed the pathogenesis of acute liver failure and the latest progress in the use of stem cell exosomes in the treatment of ALF, including the role of exosomes in inhibiting the ALF inflammatory response and regulating signal transduction pathways, the advantages of stem cell exosomes and their use as a drug‐loading system, and their pre‐clinical application in the treatment of ALF. Finally, the clinical research status of stem cell therapy for ALF and the current challenges of exosome clinical transformation are summarized.     

Bioartificial liver system based on choanoid fluidized bed bioreactor improve the survival time of fulminant hepatic failure pigs

Bioartificial liver (BAL) support system has been proposed as potential treatment method for end-stage liver diseases. We described an improved BAL system based on a choanoid fluidized bed bioreactor containing alginate-chitosan encapsulated primary porcine hepatocytes. The feasibility, safety, and efficiency of this device were estimated using an allogeneic fulminant hepatic failure (FHF) model. FHF was induced with intravenous administration of D-galactosamine. Thirty FHF pigs were divided into three groups: (1) an FHF group which was only given intensive care; (2) a sham BAL group which was treated with the BAL system with empty encapsulation, and (3) a BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. The survival times and biochemical parameters of these animals were measured, and properties of the encapsulations and hepatocytes before and after perfusion were also evaluated. Compared to the two control groups, the BAL-treated group had prolonged the survival time and decreased the blood lactate levels, blood glucose, and amino acids remained stable. No obvious ruptured beads or statistical decline in viability or function of encapsulated hepatocytes were observed. This new fluidized bed BAL system is safe and efficient. It may represent a feasible alternative in the treatment of liver failure.     

microRNA‐143‐3p attenuated development of hepatic fibrosis in autoimmune hepatitis through regulation of TAK1 phosphorylation

Autoimmune hepatitis (AIH) is a chronic liver disease due to autoimmune system attacks hepatocytes and causes inflammation and fibrosis. Intracellular signalling and miRNA may play an important role in regulation of liver injury. This study aimed to investigate the potential roles of microRNA 143 in a murine AIH model and a hepatocyte injury model. Murine AIH model was induced by hepatic antigen S100, and hepatocyte injury model was induced by LPS. Mice and AML12 cells were separated into six groups with or without the treatment of miRNA‐143. Inflammation and fibrosis as well as gene expression were examined by different cellular and molecular techniques. The model was successfully established with the elevation of ALT and AST as well as inflammatory and fibrotic markers. Infection or transfection of mir‐143 in mice or hepatocytes significantly attenuated the development of alleviation of hepatocyte injury. Moreover, the study demonstrated phosphorylation of TAK1‐mediated miRNA‐143 regulation of hepatic inflammation and fibrosis as well as hepatocyte injury. Our studies demonstrated a significant role of miRNA‐143 in attenuation of liver injury in AIH mice and hepatocytes. miRNA‐143 regulates inflammation and fibrosis through its regulation of TAK1 phosphorylation, which warrants TAK1 as a target for the development of new therapeutic strategy of autoimmune hepatitis.     

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell‐culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell‐carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1002–1012, 2013     

Comparative Proteomic Analyses of the Liver in D‐Galactosamine‐Sensitized Mice Treated with Different Toll‐Like Receptor Agonists

Acute liver failure (ALF) is a severe consequence of abrupt hepatocyte injury and has lethal outcomes. Three toll‐like receptor agonists, including polyinosinic‐polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS), and cytosine‐phosphate‐guanine (CpG) DNA, cause acute and severe hepatitis, respectively, in D‐galactosamine (D‐GalN)‐sensitized mice. However, the molecular differences among three ALF models (LPS/D‐GalN, poly(I:C)/D‐GalN, and CpG DNA/D‐GalN), are unclear. Here, tandem mass tag based quantitative proteomic analyses of three ALF mouse models are performed. 52 common differentially expressed proteins (DEPs) are identified, in three ALF groups, compared to the control. Gene ontology analyses show that among the common DEPs, ten proteins are involved in immune system process, and 39 proteins in metabolic process. Among 80,195, and 23 specifically‐expressed proteins in poly(I:C)/D‐GalN, LPS/D‐GalN, and CpG DNA/D‐GalN groups, LPS/D‐GalN‐specific proteins are mostly distributed in the endoplasmic reticulum and more enriched in metabolic pathways, whereas poly (I:C)/D‐GalN‐specific proteins are mainly in the membrane and CpG DNA/D‐GalN‐specific proteins are related to the ribosome structural composition. In conclusion, the common and specific DEPs in three ALF mouse models at molecular level are identified; and determined a close‐to‐complete reference map of mouse liver proteins which will be useful for clinical diagnosis and treatment of liver failure in humans.