Bioartificial Liver System Research Articles

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an “off-the-shelf” bioartificial liver system

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Resveralogues protect HepG2 cells against cellular senescence induced by hepatotoxic metabolites

Progressive liver disease and dysfunction cause toxic metabolites including ammonia and unconjugated bilirubin to accumulate in plasma. As the population ages alternatives to liver transplantation become increasingly important. One approach for use as a bridge to transplant or recovery is the use of bioartificial liver systems (BALS) containing primary or immortalised hepatocytes as ex-vivo replacements or supports for endogenous liver function. However, exposure to the hepatotoxic metabolites present in plasma causes the rapid failure of these cells to carry out their primary metabolic functions despite remaining viable. Hypothesizing that this loss of core hepatocyte phenotypes was caused by cell senescence we exposed HepG2 cell populations, grown in both standard two-dimensional tissue culture systems and in three dimensional cultures on novel alginate modified HEMA-MBA cryogels, to physiologically reflective concentrations of hepatotoxic metabolites and cytokines. HepG2 cells are forced into senescence by the toxic metabolites in under six hours (as measured by loss of thymidine analog incorporation or detectable Ki67 staining) which is associated with a ten to twenty-fold reduction in the capacity of the cultures to synthesise albumin or urea. This state of senescence induced by liver toxins (SILT) can be prevented by preincubation with either 2–5 µM resveratrol, its major in vivo metabolite dihydroresveratrol or a series of novel resveralogues with differential capacities to scavenge radicals and activate SIRT1 (including V29 which does not interact with the protein). SILT appears to be a previously unrecognised barrier to the development of BALS which can now be overcome using small molecules that are safe for human use at concentrations readily achievable in vivo.

Large-Scale Formation and Long-Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome-Edited GGTA1-/- Pigs for Bioartificial Liver Applications.

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1±28.1µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.

Genetically Modified Hepatocytes Targeting Bilirubin and Ammonia Metabolism for the Construction of Bioartificial Liver System.

Acute liver failure (ALF) is a complex syndrome that impairs the liver's function to detoxify bilirubin, ammonia, and other toxic metabolites. Bioartificial liver (BAL) aims to help ALF patients to pass through the urgent period by temporarily undertaking the liver's detoxification functions and promoting the recovery of the injured liver. We genetically modified the hepatocellular cell line HepG2 by stably overexpressing genes encoding UGT1A1, OATP1B1, OTC, ARG1, and CPS1. The resulting SynHeps-II cell line, encapsulated by Cytopore microcarriers, dramatically reduced the serum levels of bilirubin and ammonia, as demonstrated both invitro using patient plasma and invivo using ALF animal models. More importantly, we have also completed the 3-dimensional (3D) culturing of cells to meet the demands for industrialized rapid and mass production, and subsequently assembled the plasma-cell contacting BAL (PCC-BAL) system to fulfill the requirements of preclinical experiments. Extracorporeal blood purification of ALF rabbits with SynHeps-II-embedded PCC-BAL saved more than 80% of the animals from rapid death. Mechanistically, SynHeps-II therapy ameliorated liver and brain inflammation caused by high levels of bilirubin and ammonia and promoted liver regeneration by modulating the nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways. Also, SynHeps-II treatment reduced cerebral infiltration of neutrophils, reduced reactive oxygen species (ROS) levels, and mitigated hepatic encephalopathy. Taken together, SynHeps-II cell-based BAL was promising for the treatment of ALF patients and warrants clinical trials.

Bio-Artificial Liver Support System: A Prospective Future Therapy

Whether acute or chronic, liver failure is a state of liver dysfunction that can progress to multiorgan failure. Mortality in liver failure patients is approximately 80–90% and is caused by detoxification failure, which triggers other immediate complications, such as encephalopathy, coagulopathy, jaundice, cholestasis, and acute kidney failure. The ideal treatment for liver failure is liver transplantation, but the long waiting period for the right donor match causes unavoidable deaths in most patients. Therefore, new therapies, such as tissue engineering, hepatocyte transplantation, and stem cells, are now being studied to anticipate the patient’s condition while waiting for liver transplantation. This literature review investigated the effectiveness of some bio-artificial liver support systems using review methods systematically from international publication sites, including PubMed, using keywords, such as bio-artificial liver, acute and chronic liver failure, extracorporeal liver support system (ECLS), MARS, single-pass albumin dialysis (SPAD). Artificial and bioartificial liver systems can show specific detoxification abilities and pathophysiological improvements in liver failure patients but cannot reach the ideal criteria for actual liver function. The liver support system must provide the metabolic and synthetic function as in the actual liver while reducing the pathophysiological changes in liver failure. Aspects of safety, cost efficiency, and practicality are also considered. Identifying the technology to produce high-quality hepatocytes on a big scale is essential as a medium to replace failing liver cells. An increase in detoxification capacity and therapeutic effectiveness must also focus on patient survival and the ability to perform liver transplantation.

Establishment of a Serum-Free Hepatocyte Cryopreservation Process for the Development of an "Off-the-Shelf" Bioartificial Liver System.

To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.

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

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

HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems.

Measurement and Destruction of Porcine Endogenous Retrovirus in the Chinese Bama Minipig

Porcine hepatocytes are widely used in bioartificial liver (BAL) systems for the treatment of liver failure, and Chinese Bama minipigs (BMPs) are extensively used for animal experiments in the field of medicine in China. The genome of porcine endogenous retroviruses (PERVs) has not yet been accurately quantified, posing a threat to their clinical application because they act as a source of cells. In this study, we used genome sequence data from BMPs to predict PERV copies and their distribution. We validated and quantified the identified PERV copies and subtypes across different BMP individuals and tissues using quantitative real-timepolymerase chain reactionand droplet digital polymerase chain reaction, respectively, and found that the BMP genome contains only 11 to 21 PERV copies. Notably, they lack the C subtype, which is a relatively safe cell source. Moreover, we applied CRISPR/Cas9 technology to knock out the pol fragment of PERVs in primary renal fibroblasts (PRFs) from BMPs and obtain PERV-destructed cells. Overall, our results lay a foundation for obtaining PERV-destructed BMPs as a safe source of hepatocytes for BALs for future applications.

Bioinspired Artificial Liver System with hiPSC-Derived Hepatocytes for Acute Liver Failure Treatment.

Bioartificial liver (BAL) system has become a promising alternative to traditional liver transplantation in rescuing acute liver failure (ALF) patients. Herein, inspired by natural microstructure of hepatic lobules, a novel biomimetic bioartificial liver system (BBALS) is developed by integrating human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) -laden microparticles and semipermeable microtubes into a microfluidic platform. As the working units are hepatic lobules-like semipermeable microtubes surrounding with serum-free suspension differentiated hiPSC-Heps microcarriers, the BBALS is endowed with functional cell aggregates and effective circulation system. Thus, the BBALS possesses high cell viability, favorable function regeneration, and effective substances exchange. Based on these features, a 3D liver chip with multiple parallel BBALS units is created for filtering the plasma of ALF rabbits, which validates the research significance and application potential of the proposed BBALS. Moreover, the novel integrated BBALS is applied to treat ALF rabbits and shows great advantages in increasing survival, generating serum proteins, and decreasing inflammation. These properties point to the broad prospects of BBALS in treating related diseases and improving traditional clinical methods.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

Generation of Gene-Engineered Human Hepatoma Cells with Heat-Inducible Liver Functions

Hepatoma cells derived from liver carcinoma are a candidate cell source for bioartificial liver (BAL) systems due to their high proliferative capacity, although liver function of hepatoma cells is considerably low compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells with inducible high liver function were established by transducing liver-enriched transcription factor (LETF) genes. In this study, we aimed to develop new gene-engineered human hepatoma cells, in which high liver functions are inducible by heat treatment. For this purpose, we constructed a gene expression system for eight LETF genes under control of tetracycline-dependent transactivator (tTA), and the system was introduced into the genome of HepG2-HSP cells, in which a tTA expression system induced by a heat-shock protein promoter with transcriptional amplification was introduced into HepG2 cells. Thus, the heat-inducible tTA promotes LETF genes to induce liver function. Upon the heat treatment of the cells (HepG2-HSP/8F) at 43°C for 30 min, liver functions such as albumin secretion and cytochrome P450 were significantly enhanced. The cells with heat-inducible liver function can be used as a new cell source for various hepatic studies including construction of BAL systems.

Hollow fibre membrane bioreactor functionalized with GO‐cryogel 3D matrix promotes liver cell anchoring and their functional maintenance

AbstractThe liver support system (bioartificial liver, BAL) is a potentially efficient treatment during reversible acute liver disease or while waiting for a liver transplant. The main challenge in implementing BAL is a bioreactor, which can fulfil the needs of hepatocyte culture, mass transfer and immunobarriers. In the present study, polymer nanocomposite hollow fibre membranes (HFMs) were fabricated using polyethersulphone (P) as a base polymer, vitamin E TPGS (T) as an additive and carboxylated carbon nanotubes (C) as a filler. These PTC HFMs were functionalized with cryogel (Cr), with or without carboxylated graphene oxide (G), known as PTC‐Cr or PTC‐CrG HFMs, respectively. These HFM substrates were characterized and evaluated for hemocompatibility, cytotoxicity and cellular functionality. Among all the HFM substrates, PTC‐CrG HFMs showed superior hemocompatibility and minimal cytotoxicity. Furthermore, a liver cell bioreactor was developed and studied using PTC‐CrG HFMs. The goat primary liver cells were seeded in a bioreactor and cultured in continuous mode. The cell functionality was better maintained in the continuous mode as compared to that in the batch culture. The three‐dimensional PTC‐CrG HFMs‐based bioreactor can be used for liver cell bioreactor, bioartificial liver system, drug testing/toxicology metabolic study and mass cell culture.

Enhanced cellular functions of hepatocytes in the hyaluronate-alginate-chitosan microcapsules.

The study aimed to develop a biocompatible microcapsule for hepatocytes and create a bio-mimic microenvironment for maintaining hepatic-specific functions of hepatocytes in vitro. The work is proposed for the bioartificial liver system in the treatment of liver failure. In this study, microcapsules were prepared with hyaluronate (HA)/sodium alginate (SA) as an inner core and an outer chitosan (CS) shell via one-step spraying method. C3A cells were encapsulated in microcapsules to examine the biocompatibility of HA-SA-CS microcapsules. MTT and fluorescence microscopy indicated that C3A cells had high viability in the HA-SA-CS microcapsules. The liver-specific functions, such as urea and albumin synthesis, and CYP1A2 and CYP3A4 activities from encapsulated cells were increased in the HA-SA-CS microcapsules compared to the SA-CS microcapsules. The gene expressions of CYP450 related genes were also increased by HA on day 3. The study suggests that HA-SA-CS microcapsules have good biocompatibility and can maintain a favorable environment for hepatocytes. This approach has improved the preservation of liver cells' metabolic functions and could be a candidate for the bioartificial liver system.

Beneficial bioartificial livers

Bioengineering Bioartificial livers are an attractive option as a bridge to transplant or to promote liver regeneration in cases of acute liver failure. Li et al. tested an extracorporeal bioartificial liver system composed of human liver progenitor-like cells cultured on macroporous scaffolds in a bioreactor that provides alternating air-liquid exposure. Three hours of treatment improved survival, reducing inflammation and promoting native liver regeneration in pigs with drug-induced acute liver failure. These results suggest that extracorporeal, cell-based bioartificial livers may be a promising treatment for acute liver failure. Sci. Transl. Med. 12 , eaba5146 (2020).

Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD).We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation.These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.

Fabrication and in vitro evaluation of a packed-bed bioreactor based on galactosylated poly(ethylene terephthalate) microfibrous scaffolds

Bioartificial liver (BAL) comprising a functional bioreactor represents a promising treatment, bridging patients with acute liver failure (ALF) to liver transplantation. In this research, a packed-bed (PB) bioreactor by utilizing galactosylated poly(ethylene terephthalate) microfibrous carrier (PET-Gal) was fabricated and evaluated in vitro. Human induced hepatocytes (hiHeps) were dynamically seeded into a PET-Gal loaded bioreactor and cultured for 10 days. During perfusion culture, the effects of bioreactor scaffold on hepatocyte seeding, growth and functionality (albumin secretion and urea production) were determined, compared with that of control with non-modified PET carrier. Afterwards, drug metabolism and detoxification ability of hiHeps were measured. Results demonstrated that the seeding rate and proliferation fold within PET-Gal loaded bioreactor were significantly higher than that of control, resulting in a cell density of (8.17 ± 0.52)×107 cells/cm3. In contrast to flattened morphology observed in the control, hiHeps cultured in PET-Gal based bioreactor displayed as three-dimensional (3D) cell aggregates with close cell-matrix and cell-cell interactions and high cell viability, leading to higher albumin secretion and urea production. Besides, there were high levels of phase I drug metabolism and ammonia elimination for PET-Gal cultured cells. Therefore, the PET-Gal based PB bioreactor fabricated herein supports high-density hepatocyte growth and meanwhile preserves hepatic functionality, showing great potential in BAL systems application.

SOD2 promotes the expression of ABCC2 through lncRNA CLCA3p and improves the detoxification capability of liver cells

Superoxide dismutase 2 (SOD2) is a key enzyme for scavenging reactive oxygen species produced by mitochondria, which plays an important role in maintaining cellular homeostasis. However, its effects on the detoxification capability of liver cells have not been reported. In this study, we found that change in SOD2 expression affects the proliferation of liver cells. Genome-wide microarray analysis showed that SOD2 positively regulates the drug transporter ABCC2, and co-expression analysis suggested that lncRNA CLCA3P participates in the process. Further experiments showed that SOD2 can promote the expression of CLCA3P, which increases the transcription of ABCC2 by interacting with the transcription factor IRF1. By increasing ABCC2 expression SOD2 facilitates drugs efflux of liver cells and thus promotes their survival under a drug-toxic environment. This study elucidates the improvement of the detoxification of liver cells by a regulatory axis, SOD2-CLCA3P-IRF1-ABCC2, and provides novel insight into the modification of human liver cells that can be applied to bioartificial liver system or the study of SOD2 in drug metabolism.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma

BackgroundFor its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. MethodsC3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. ResultsCell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. ConclusionsC3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

Human hepatocellular adenoma as a promising cell source for bioartificial liver systems.

The present study assessed human hepatocellular adenoma (HCA) as a potential source of biological material for bioartificial liver (BAL) systems. The histological characteristics of HCA tissues from 8 patients were examined using hematoxylin and eosin staining. The glycogen synthesis capacity of HCA cells was assessed using Periodic Acid-Schiff (PAS) staining and the expression of genes involved in liver function were examined using immunohistochemical staining (IHC) and reverse transcription-quantitative PCR analysis. Primary cells from HCA tissues were subsequently isolated and cultured in vitro. Cells within HCA tissues from 8 patients exhibited a polygonal shape, similar to that of cells in adjacent normal liver tissues. PAS staining of HCA tissues indicated the capacity of these cells to synthesize and store glycogen. Furthermore, IHC and PCR analyses revealed that the expression of liver function genes in HCA tissues were similar to those observed within normal adjacent liver tissues. Primary cells isolated from HCA tissues exhibited an irregular polygonal shape and positive in vitro growth. The current study demonstrated that HCA tissues exhibit histological and functional characteristics matching those of normal human liver tissue and may therefore be a promising alternative to hepatocytes as a source of biological material for BAL systems.