Bioartificial Liver Support System Research Articles

Functional hit 1 (FH1)-based rapid and efficient generation of functional hepatocytes from human mesenchymal stem cells: a novel strategy for hepatic differentiation.

BackgroundBecause the liver is central to the physiology of the body, primary hepatocytes are widely used in liver pathology and physiological research, such as liver drug screening, bioartificial liver support system, and cell therapy for liver diseases. However, the source of primary hepatocytes is limited. We describe a novel non-transgenic protocol that facilitates the rapid generation of hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), providing a new source of functional hepatocytes.MethodsIn this study, we used hUC-MSCs and human induced pluripotent cells (iPSCs) derived mesenchymal stem cells (iMSCs) to investigate the new induction strategy. Passage 3 MSCs were induced into hepatocyte-like cells using small-molecule compounds combined with cell factors in vitro. Functional hit 1 (FH1), a promising small molecule compound was achieved to replace HGF in the hepatocyte maturation stage to induce the hepatocyte-like cells differentiation.ResultsWe rapidly induced hUC-MSCs and human iMSCs into hepatocyte-like cells within 10 days in vitro, and the cells were morphologically similarly to both hepatocytes derived from the hepatocyte growth factor (HGF)-based method and the primary hepatocytes. They expressed mature hepatocyte special genes and achieved functions such as glycogen storage, albumin expression, urea secretion, cytochrome P450 activity, Low-density lipoprotein (LDL) uptake, and indocyanine green (ICG) uptake.ConclusionsWe successfully established a small-molecule protocol without using HGF to differentiate MSCs into hepatocyte-like cells, which provides a rapid and cost-effective platform for in vitro studies of liver disease.

A bioartificial liver support system integrated with a DLM/GelMA-based bioengineered whole liver for prevention of hepatic encephalopathy via enhanced ammonia reduction.

Although bioartificial liver support systems (BLSSs) play an essential role in maintaining partial liver functions and detoxification for liver failure patients, hepatocytes are unanimously seeded in biomaterials, which lack the hierarchal structures and mechanical cues of native liver tissues. To address this challenge, we developed a new BLSS by combining a decellularized liver matrix (DLM)/GelMA-based bioengineered whole liver and a perfusion-based, oxygenated bioreactor. The novel bioengineered whole liver was fabricated by integrating photocrosslinkable gelatin (GelMA) and hepatocytes into a DLM. The combination of GelMA and the DLM not only provided a biomimetic extracellular microenvironment (ECM) for enhanced cell immobilization and growth with elevated hepatic functions (e.g., albumin secretion and CYP activities), but also provided biomechanical support to maintain the native structure of the liver. In addition, the perfusion-based, oxygenated bioreactor helped deliver oxygen to the interior tissues of the bioengineered liver, which was of importance for long-term culture. Most importantly, this new bioengineered whole liver decreased ammonia concentration by 45%, whereas direct seeding of hepatocytes in a naked DLM showed no significant reduction. Thus, the developed BLSS integrated with the DLM/GelMA-based bioengineered whole liver can potentially help elevate liver functions and prevent HE in liver failure patients while waiting for liver transplantation.

Transition of the Field from Biochemical Engineering to Pharmaceutical Sciences during 40 Years of the Research

This review reflects back over almost 40 years of the author's basic research conducted at Graduate School of Pharmaceutical Sciences, Osaka University, Japan. After performing postdoctoral research in USA, the author became a research associate at Prof. Yoshiharu Miura's lab and started research on Biochemical Engineering in 1984. At that time, the main research purpose was to solve global environmental issues for maintaining human health. The author's achievements included novel useful material production system under inorganic conditions and genetically engineered whole-cell bacterial sensors detecting arsenite by naked eye without a detecting device. Another theme in the lab was to construct bioartificial liver support system. Various scaffolds for hepatocytes were newly prepared for constructing the compact reactor. Besides the bioreactor study, the author conducted cell transplantation research for the treatment of chronic liver diseases. It was shown that mesenchymal stem cells derived from third molars (wisdom teeth) could differentiate into hepatocytes and exhibit therapeutic effects in liver-damaged animals. After 2006, the lab started research on drug delivery systems, including noninvasive delivery of drugs such as peptides and nucleic acids by regulating epithelial tight junctions. Many substances enabling drug delivery through "paracellular" route were newly prepared. The author started basic research on Biochemical Engineering in the 1970s. Although these studies eventually shifted into the pharmaceutical field, the underlying concept was based on "engineering" throughout a 40-year research period. The author cordially thanks all colleagues for supporting engineering research in our lab.

Evaluation of the mass transfer rate using computer simulation in a three-dimensional interwoven hollow fiber-type bioartificial liver.

To determine the most efficient design of a hollow fiber-based bioreactor device for a bioartificial liver support system through comparative bioengineering evaluations. We compared two types of hollow fiber-based bioreactors, the interwoven-type bioreactor (IWBAL) and the dialyzer-type bioreactor (DBAL), by evaluating the overall mass transfer coefficient (K) and the convective coefficient (X). The creatinine and albumin mass transfer coefficients and convective coefficients were calculated using our mathematical model based on the homoporous theory and the modified Powell method. Additionally, using our model, we simulated the mass transport efficiency in clinical-scale BALs. The results of this experiment demonstrate that the mass transfer coefficients for creatinine and albumin increased proportionally with velocity with the IWBAL, and were consistently greater than that found with the DBAL. These differences were further enhanced in the simulation of the large-scale model. Our findings indicate that the IWBAL with its unique 30° cross hollow fiber design can provide greater solute removal and more efficient metabolism when compared to the conventional DBAL design.

1. Development of bioengineered xenogeneic decellularized humanized liver for the development of extracorporeal liver for the treatment of acute liver failure

Background and Aims: Acute as well as chronic liver failures are major fatal problems, which lead up to 60–80% mortality every year. In acute condition, systemic inflammation and accumulation of toxic compounds (ammonia) in liver results in multi-organ failure which leads to hepatic encephalopathy. Liver transplantation is limited due to timely unavailability of enough donors, post-transplantation complications and high cost involvement. The present study demonstrates a promising strategy of using bioartificial extracorporeal liver support system prepared through decellularization and repopulation of xenogenic liver with human hepatic progenitor cells (hHPCs). Methods: Xenogenic liver was decellularized by perfusion method using retrograde change of detergents and other chemicals. Removal of nuclear components, retention of ECM and vascular integrity of whole decellularized liver was identified. DiD-labeled hHPCs were infused to identify the cells distribution and engraftment efficiency. Repopulation of hHPCs within the decellularized liver scaffold was determined by SEM analysis whereas the functional activity of repopulated cells was determined by ammonia detoxification experiment by urea quantification. Results: The decellularized liver vascular network was able to withstand fluid flow that entered through a central inlet vessel, branched into an extensive capillary bed, and coalesced into a single outlet vessel. Repopulated hHPCs showed homogeneous distribution within the decellularized liver scaffold showing defined vascular tree with multiple branching and residual niches of proliferating cells. Ammonia detoxification showed the functional activity and future applicability of repopulated humanized liver scaffold as extra-corporeal natural organ support system. Conclusions: This study demonstrate an innovative technology for bioengineering humanized extra- corporeal liver system as better approach for ammonia detoxification as temporary support to the failing liver. The authors have none to declare.

Co-culture system of hepatocytes and endothelial cells: two in vitro approaches for enhancing liver-specific functions of hepatocytes.

Although hepatocyte transplantation and bioartificial liver support system provide new promising opportunities for those patients waiting for liver transplantation, hepatocytes are easily losing liver-specific functions by using the common in vitro cultured methods. The co-culture strategies with mimicking the in vivo microenvironment would facilitate the maintenance of liver-specific functions of hepatocytes. Considering that hepatocytes and endothelial cells (ECs) account for 80-90% of total cell populations in the liver, hepatocytes and ECs were directly co-cultured with hepatic stellate cells (HSCs) or adipose tissue-derived stem cells (ADSCs) at a ratio of 700:150:3 or 14:3:3 in the present study, and the liver-specific functions were carefully analyzed. Our results showed that the two co-culture systems presented the enhanced liver-specific functions through promoting secretion of urea and ALB and increasing the expressions of ALB, CYP3A4 and HNF4α, and the vessel-like structure in the co-culture system consisted of hepatocytes, ECs and ADSCs. Hence, our results suggested that the directly co-culture of hepatocytes and ECs with HSCs or ADSCs could significantly improve liver-specific functions of hepatocytes, and the co-culture system could further promote angiogenesis of ECs at a later stage. Therefore, this study provides potential interesting in vitro strategies for enhancing liver-specific functions of hepatocytes.

Phase 1/2a trial of a bioartificial liver support system for acute liver failure patients

Phase 1/2a trial of a bioartificial liver support system for acute liver failure patients

Progress in hepatocyte status detection and its application in bioartificial liver support system

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.

Functionally coated polyethersulfone hollow fiber membranes: A substrate for enhanced HepG2/C3A functions.

Hollow fiber membrane (HFM) based liver assist systems are a life-saving bridge for patients until a donor organ is available for transplantation or until liver regeneration. However, liver cell attachment and functional maintenance on HFM surface is a major challenge in bio-artificial liver (BAL) support systems. In the present study, novel glutaraldehyde (GTA)-crosslinked gelatin (gel)-coated polyethersulfone (X-gel-PT) HFMs were manufactured using triple orifice spinneret by the dry-wet spinning method. HFMs were characterized for morphology, outer surface roughness, hydrophilicity, tensile strength, thermal stability, BET surface area and pore volume measurements, permeability and rejection. Fourier transform infrared spectroscopy, and transmission electron microscopy confirmed the GTA-crosslinked gel-coating in the X-gel-PT HFMs, which provided the desirable extracellular matrix-like environment to the HepG2/C3A cells. The results of in-vitro hemocompatibility tests showed the better suitability of the developed HFMs for the blood-contact application. X-gel-PT HFMs showed significantly better cellular attachment and proliferation of HepG2/C3A cells on day 3 and 6, as shown by scanning electron and confocal microscopy. Significantly high urea synthesis and albumin secretion seen indicated the improved functional and metabolic activity of HepG2/C3A cells. Thus, the developed X-gel-PT HFMs is a suitable substrate for the hepatocyte culture, mass culture, and development of BAL support system.

Effects of gelling bath on the physical properties of alginate gel beads and the biological characteristics of entrapped HepG2 cells.

Optimizing alginate gel beads is necessary to support the survival, proliferation, and function of entrapped hepatocytes. In this study, gelling bath was modified by decreasing calcium ion concentration and increasing sodium ion concentration. Alginate gel beads (using 36% G sodium alginate) prepared in the modified gelling bath had more homogeneous structure and better mass transfer properties compared with the traditional gelling bath that contains only calcium ions. Moreover, alginate gel beads generated in the modified gelling bath could significantly promote the HepG2 cell proliferation and the growth of cell spheroids, and maintain the albumin secretion ability similar to alginate gel beads prepared in the traditional gelling bath with only calcium ions. The mass transfer properties and cell proliferation were similar in ALG beads with different M/G ratio (36% G and 55% G) generated in the modified gelling bath, whereas they were significantly increased compared with alginate gel beads (55% G) in traditional gelling bath. These results indicated that adjusting the gelling bath was a simple and convenient method to enhance the mass transfer properties of alginate gel beads for 3D hepatocyte culture, which might provide more hepatocytes for the bioartificial liver support system.

Functional Evaluation of a Bioartificial Liver Support System Using Immobilized Hepatocyte Spheroids in a Porcine Model of Acute Liver Failure

Bioartificial livers (BAL) may offer acute liver failure (ALF) patients an opportunity for cure without liver transplantation. We evaluated the efficacy of a spheroid-based BAL system, containing aggregates of porcine hepatocytes, in a porcine model of ALF. ALF pigs were divided into three groups. The control group consisted of treatment naïve pigs (n = 5), blank group consisted of pigs that were attached to the BAL system not containing hepatocytes for 12 hours (n = 5) and BAL group consisted of pigs that were attached to the BAL containing hepatocytes for 12 hours (n = 5). Increase in serum ammonia levels were significantly greater in the blank group (P < 0.01) and control group (P < 0.01), compared to the BAL group during the treatment period. Increase in ICP was significantly greater in the control group compared to the BAL group (P = 0.01). Survival was significantly prolonged in the BAL group compared to the blank group (P = 0.03). A BAL system with a bioreactor containing hepatocyte spheroids showed effective clearance of serum ammonia, preservation of renal function and delayed ICP increase in a porcine model of ALF.

Induction of hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells by defined microRNAs.

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl4‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.

Preliminary Research of Off-Line Bioartificial Liver on Patients with Hbv Related Acute-On-Chronic Liver Failure

Objective: The study aims to construct an off-line bioartificial liver support system (off-line BAL) with human liver cell line, and explore it’s safety and effect in patients suffering with HBV-related acute on chronic liver failure (ACLF). Methods: The off-line BAL was constructed with cultured HepG2 cell. Twenty patients with HBV-related ACLF were randomly separated into the two groups. Patients in the treatment group were dealt with plasma exchange (PE) first and then BAL treatment. The control group received a therapy of PE only . The clinical parameters were assessed at different times and survival rate was evaluated at 3 months. Results: In the treatment group, 9 patients’ general conditions and clinical symptoms were improved, total bilirubin decreased about 44.27%, MELD scores decreased to 21.71 from 24.26, prothrombin activity (PTA) increased to 48.97%, and there was a significant difference between pretreatment and post-treatment. Compared to the control group, PTA increased dramatically (51.02% vs. 37.24%; P=0.0477) at 4 weeks and MELD score decreasedsignificantly (21.71 vs. 24.47; P=0.0409) at post-treatment in BAL groups. During the 12 weeks, the survival rates were 70% and 50% (P=0.3613) in the treatment and control groups. No severe adverse events occurred and no liver tumor was found following three years of observation. Conclusions: The off-line BAL may be safe for patients with liver failure. It can improve the patients’ clinical conditions and laboratory parameters, but it has no obvious benefit compared to PE treatment. The routine clinical application still needs further evidence.

Fabrication of macroporous cryogels as potential hepatocyte carriers for bioartificial liver support

Two different cryogels composed of copolymer of acrylonitrile (AN) and N-vinyl-2-pyrrolidone (NVP) (poly(AN-co-NVP)) and interpenetrated polymer networks (IPN) of chitosan and poly(N-isopropylacrylamide) (poly(NiPAAm)-chitosan) were fabricated by gelation at sub-zero temperatures. The two cryogels possess an interconnected network of macropores of size 20–100μm and efficient transport properties as determined by physiochemical analysis. Both cryogels support in vitro growth and function of fibroblasts (COS-7) and human liver hepatocarcinoma cells (HepG2). The cryogels are hemocompatible as demonstrated by low albumin adsorption and platelet adherence. Furthermore, in vivo implantation of poly(NiPAAm)-chitosan cryogel in mice shows its biocompatibility with the surrounding tissue. Primary rat hepatocytes grown on poly(NiPAAm)-chitosan cryogel for 96h formed cellular aggregates and maintained their functions in terms of, ammonia removal, ureagenesis and drug detoxification. Cryogel-based closed continuous bioreactor systems could maintain HepG2 cells at high density for 7 days. Off-line clinical evaluation of these cryogel-based bioreactors showed the ability of immobilized cells to detoxify circulating plasma obtained from patients with acute on chronic liver failure (ACLF). Altogether, the presented data suggests cryogels as a potential bioreactor matrix for bio-artificial liver support system.

Simulation and experimental research on micro-channel for detecting cell status in bio-artificial liver.

Bioartificial liver support system (BALSS) based on culturing hepatocytes is an important research field for the treatment of acute liver failure. It is necessary to monitor the state of liver cell functions during the treatment of BALSS in order to guide clinical treatment. To design a micro-channel chip to achieve flash mixing for timely detection of liver cell status in bioreactors and improving liver cells growth environment to ensure the efficacy of the bio-artificial liver support system. Alanine aminotransferase (ALT) and Urea are chosen as detection indicators to reflect the degree of liver cell injury and the detoxification function. A diamond tandem structure micro-channel is designed and optimized to achieve the efficient mixing of serum and ALT or Urea reagent. The simulation and experimental results show that the diamond tandem structure micro-channel can significantly improve the mixing efficiency and meet the online detecting requirements. The easily controllable diamond tandem structure micro-channel combines the advantages of active and passive mixer and can effectively mix the serum and ALT or Urea reagent. It lays the foundation for online monitoring of liver cells and will help to improve the viability of liver cell in the bioreactor.

P1309 : A randomized, open-label, multicenter, controlled study to assess safety and efficacy of ELAD® human cell-based bio-artificial liver support system (ELAD®) in subjects with alcohol-induced liver decompensation (AILD)

P1309 : A randomized, open-label, multicenter, controlled study to assess safety and efficacy of ELAD® human cell-based bio-artificial liver support system (ELAD®) in subjects with alcohol-induced liver decompensation (AILD)

Development of a New Bioartificial Liver Support System Using a Radial-flow Bioreactor

Development of a New Bioartificial Liver Support System Using a Radial-flow Bioreactor

Protein Content Assessment During Treatment With the ELAD Bioartificial Liver Support System.

Protein Content Assessment During Treatment With the ELAD Bioartificial Liver Support System.

Establishment of cytochrome P450 3A4 and glutathione S-transferase A1-transfected human hepatoma cell line and functional analysis.

This study aimed to enhance the drug metabolism function of the human hepatoma cell line C3A and to explore the related significance for patients with severe liver disease. The important liver phase I and phase II drug metabolism enzymes, cytochrome P450 3A4 (CYP 3A4) and glutathione S-transferase A1 (GST A1), were constructed into a double expression vector and then transfected into C3A cells. Furthermore, in order to increase the expression of CYP 3A4 and GST A1, they were optimized according to human optimal codons. Another double-expression vector, pBudCE4.1-optimized CYP 3A4-optimized GST A1, was constructed and then transfected into C3A to establish a stable cell line. The drug metabolism function of C3A was evaluated. Sequence determination and analysis results showed that the recombinant plasmid pBudCE4.1-CYP 3A4-GST A1 met the application standard and its transfection was successful. The expression and activity of CYP 3A4 and GST A1 in unoptimized C3A cells were higher than those in blank C3A cells. Unoptimized C3A had a better drug metabolism function. Although some C3A cells transfected with pBudCE4.1-optimized CYP 3A4-optimized GST A1 survived, they grew slowly, and were therefore not applicable in clinical practice. Unoptimized C3A is superior to blank C3A in drug metabolism, and could be applied in the bioartificial liver support system as a new material.

Optimization of Oxygenation Parameters in Bioreactor Based on Oxygen Carrier

In bioartificial liver support system, based on the high density cell culture, the key point is to provide appropriate dissolved oxygen(DO) concentration for hepatocytes in bioreactor. So in order to improve the hypoxia environment, oxygen carrier was introduced to enhance the oxygenation in the bioreactor, which is of great importance. In this paper, the demand of oxygen carriers and the oxygenation rate after the adjustment of inlet velocity, the oxygen pressure and other parameters were studied through experiments. At last, the optimized oxygenation parameters were obtained.