Inventing Engineered Organoids for end-stage liver failure patients.

End-stage liver disease (ESLD) is a term used clinically in reference to a group of liver diseases with liver transplantation as the choice of treatment. Due to the limitations of liver transplantation, alternative treatments are needed. The use of primary human hepatocytes represents a valid alternative treatment, but the limitations related to hepatocyte quality, viability, function, conservation, and storage need to be overcome. Transplanted hepatocytes have only been followed for 6-9 months. Therefore, long-term causes of failures are not yet established, including rejection, apoptosis, or other causes. Other alternative therapies to replace liver transplantation include plasmapheresis, hemodiafiltration, and artificial livers. Unfortunately, these methods are highly limited due to availability, high cost, anaphylaxis reaction, development-deposition of immune-complexes, and restricted functionality. Liver organoids, which utilize stem cells instead of 'impractical' adult hepatocytes, may be a solution for the development of a complex bioartificial liver. Recent studies have explored the benefits of differentiating mature hepatocytes from stem cells inside a bioreactor. When the use of human-induced Hepatocytes (hiHeps) was investigated in mouse and pig models of liver failure, liver failure markers were decreased, hepatocyte function indicated by albumin synthesis improved, and survival time increased. Bioartificial liver treatment may decrease the infiltration of inflammatory cells into liver tissue by down-regulating pro-inflammatory cytokines.

Dimethyl sulfoxide for cryopreservation of alginate encapsulated liver cell spheroids in bioartificial liver support; assessments of cryoprotectant toxicity tolerance and dilution strategies.

The Bioartificial Liver (BAL) is an extra-corporeal liver support designed to support the function of the Liver in patients with impaired liver function. The BAL biomass consists of alginate encapsulated liver spheroids (AELS). To facilitate rapid delivery of a BAL to patients the AELS are cryopreserved using a DMSO-containing cryoprotectant solution. This study assesses toxicity of DMSO in AELS at concentrations and temperatures relevant to the cryopreservation and recovery process of a cellular biomass. Additionally, it develops a process to remove DMSO from AELS before delivery of cell product to patients. Exposure of AELS to DMSO, at a concentration of 12% (v/v) for 10 min did not have a negative effect on the viability of the AELS up to 24 h after exposure, irrespective of the exposure temperature between 37 C and 0 C. Evidence of toxicity was only seen with exposure to 40% (v/v) DMSO, which was more notable at warm temperatures. Post-Thaw removal of DMSO was measured by determining the DMSO concentration of the post-thaw washes using refractometry. Washing AELS 3 times in tapering concentrations of Glucose supplemented DMEM at an AELS:wash ratio of 1:2 was sufficient to reduce DMSO to undetectable levels (<1%). The study demonstrated that the thawing method minimised DMSO toxicity to the BAL biomass, and the post-thaw washing protocol successfully removed all the DMSO present in the cryopreserved BAL. Thereby enabling effective cryopreservation of the BAL for future clinical translation.

Fructus Psoraleae-Induced Severe Liver Injury and Treatment With Two Artificial Liver Support Systems: A Case Series Study.

To describe the clinical features and outcomes of patients with suspected Fructus Psoraleae (FP)-induced severe liver injury who underwent treatment with two artificial liver support systems (ALSSs). The cases of 12 patients with severe liver injury by FP were enrolled. We evaluated the tolerability of, and changes in biochemical parameters after treatment with plasma exchange combined with hemofiltration and double plasma molecular absorption system, and 6-month follow-up information were collected. The median age of the 12 patients was 60 years and nine (75%) patients were females. All patients had jaundice as the initial symptom. Two ALSS types were used to treat the patients. The group that underwent plasma exchange combined with hemofiltration showed remarkable improvements in ALT, AST, total bilirubin (TB), GGT and international normalized ratio levels (AST, TB, international normalized ratio, P < 0.01; ALT, GGT, P < 0.05), and the levels of AST, ALP, TB, and total bile acid decreased significantly in the double plasma molecular absorption system group after treatment (TB, P < 0.01; AST, ALP, total bile acid P < 0.05). During 6 months of follow-up, two patients died, two became chronic, and eight recovered to normal. FP can cause clinically severe liver injury, characterized by gastrointestinal symptoms and jaundice, which can lead to death or become chronic. Both ALSSs were safe and well tolerated in drug-induced liver injury patients. After ALSS treatment, the levels of biochemical indicators of liver function improved significantly, indicating that ALSS might be beneficial for patients with severe drug-induced liver injury.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Clinical translation of bioartificial liver support systems with human pluripotent stem cell-derived hepatic cells.

There is currently a pressing need for alternative therapies to liver transplantation. The number of patients waiting for a liver transplant is substantially higher than the number of transplantable donor livers, resulting in a long waiting time and a high waiting list mortality. An extracorporeal liver support system is one possible approach to overcome this problem. However, the ideal cell source for developing bioartificial liver (BAL) support systems has yet to be determined. Recent advancements in stem cell technology allow researchers to generate highly functional hepatocyte-like cells from human pluripotent stem cells (hPSCs). In this mini-review, we summarize previous clinical trials with different BAL systems, and discuss advantages of and potential obstacles to utilizing hPSC-derived hepatic cells in clinical-scale BAL systems.

Engineered Liver Platforms for Different Phases of Drug Development.

Drug-induced liver injury (DILI) remains a leading cause of drug withdrawal from human clinical trials or the marketplace. Owing to species-specific differences in liver pathways, predicting human-relevant DILI using in vitro human liver models is crucial. Microfabrication tools allow precise control over the cellular microenvironment towards stabilizing liver functions for weeks. These tools are used to engineer human liver models with different complexities and throughput using cell lines, primary cells, and stem cell-derived hepatocytes. Including multiple human liver cell types can mimic cell-cell interactions in specific types of DILI. Finally, organ-on-a-chip models demonstrate how drug metabolism in the liver affects multi-organ toxicities. In this review we survey engineered human liver platforms within the needs of different phases of drug development.

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

PURPOSE: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. METHODS: To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10⁷ hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. RESULTS: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. CONCLUSION: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

A human liver microphysiology platform for investigating physiology, drug safety, and disease models.

This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 µM troglitazone or 210 µM nimesulide produced acute toxicity within 2-4 days, whereas 28 µM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.

Current and future directions in the treatment and prevention of drug-induced liver injury: a systematic review.

While the pace of discovery of new agents, mechanisms and risk factors involved in drug-induced liver injury (DILI) remains brisk, advances in the treatment of acute DILI seems slow by comparison. In general, the key to treating suspected DILI is to stop using the drug prior to developing irreversible liver failure. However, predicting when to stop is an inexact science, and commonly used ALT monitoring is an ineffective strategy outside of clinical trials. The only specific antidote for acute DILI remains N-acetylcysteine (NAC) for acetaminophen poisoning, although NAC is proving to be beneficial in some cases of non-acetaminophen DILI in adults. Corticosteroids can be effective for DILI associated with autoimmune or systemic hypersensitivity features. Ursodeoxycholic acid, silymarin and glycyrrhizin have been used to treat DILI for decades, but success remains anecdotal. Bile acid washout regimens using cholestyramine appear to be more evidenced based, in particular for leflunomide toxicity. For drug-induced acute liver failure, the use of liver support systems is still investigational in the United States and emergency liver transplant remains limited by its availability. Primary prevention appears to be the key to avoiding DILI and the need for acute treatment. Pharmacogenomics, including human leukocyte antigen genotyping and the discovery of specific DILI biomarkers offers significant promise for the future. This article describes and summarizes the numerous and diverse treatment and prevention modalities that are currently available to manage DILI.

Hepatocyte Isolation, Culture, and Its Clinical Applications / 한양의대학술지

Hepatocytes, parenchymal cells of the liver, are specially differentiated cells to perform most of the body metabolisms. Many clinicians are interested in utilizing hepatocytes as cell therapeutics. A great number of investigators have been harvesting hepatocytes using two-step portal vein perfusion method, in which Ca2+-free EDTA-containing buffer and Ca2+-enriched collagenase solution are pumped into liver in sequence. Among various attempts for long-term culture of hepatocytes, collagen gel sandwich configuration is recognized to be the most effective technique. In the biomedical field, hepatocytes have been used in three methods of applications. First is hepatocyte transplantation for the treatment of acute, chronic liver failure and metabolic diseases. Donated livers not suitable for organ transplantation are rare, which is the major human hepatocyte source. This shortage of human hepatocyte source is expected to be resolved by virtue of rapid progressing stem cell technologies. The second application is biological components of bioartificial liver (BAL) system for acute liver failure patients. Due to the lack of functional activity of clinically studied BAL systems and difficulty of establishing a manufacturing system for ready-to-use, additional research activities are stagnated. The third utilization of hepatocytes is in vitro drug screening studies such as drug metabolism, transport, biliary excretion, and toxicity tests. If cell therapeutic treatments using hepatocytes are clinically valuable to some types of liver diseases, the demand for liver transplantation would be significantly diminished.

Effect of fulminant hepatic failure porcine plasma supplemented with essential components on encapsulated rat hepatocyte spheroids.

The development of bioartificial liver (BAL) systems has required detailed information about the functional capabilities of cultured hepatocytes during blood or plasma passage. In this study we investigated the effects of porcine plasma and various supplements on the viability and function of adult rat hepatocytes in vitro. Primary rat hepatocytes cultured in porcine plasma supplemented with various substances showed albumin synthesis rates and viability equal to or higher than those of controls. Supplementation with calcium chloride, magnesium sulfate, trace elements, amino acids, insulin, and epidermal growth factor were essential to maintain viability and high albumin synthesis. Especially, trace elements showed significantly higher and longer albumin secretion. Isolated rat hepatocytes were cultured in Spinner flasks for 24 hours to form spheroids that were harvested and encapsulated with chitosan-alginate solution before transfer to the bioreactor in the BAL system. Encapsulated rat hepatocyte spheroids cultured with porcine plasma including trace elements showed higher viability (57%) than controls (40%) after 24 hours, with ammonia removal values of 30.92 µg/10(6) cells versus the control 9.04 µg/10(6) cells. After 24 hours of operation the urea secretion value of encapsulated rat hepatocyte spheroids cultured in porcine plasma in the presence versus absence of trace elements was 76.73 µg/10(6) cells and 18.80 µg/10(6) cells, respectively. We concluded that encapsulated hepatocyte spheroids in a packed-bed bioreactor operated with human plasma including trace elements enhanced cell viability and liver function as a bases for an in vivo clinical trial of the BAL system.

Improved preclinical safety assessment using micro-BAL devices: the potential impact on human discovery and drug attrition.

Hepatotoxicity is often unpredictable in the early phase of drug discovery and leads to drug attrition in preclinical and clinical development. Here, we discuss the conventional preclinical liver models that do not mimic in vivo livers. We focus on key components such as new sources of hepatocyte-derived human stem cells, enhanced direct oxygenation, defined biocompatibility nanoscaffolds, organotypical cellular models, dynamic culture, and metabolite status inside and outside the cell for effective configuration for the development of a bioartificial liver (BAL) device to mimic the in vivo liver microenvironment. The potential for development of BAL devices could open up new avenues in: (i) hepatotoxicity assessment for selecting drug candidates during preclinical screening; and (ii) therapeutic approaches for liver cell therapy at the clinical stage.

What clinical alternatives to whole liver transplantation? Current status of artificial devices and hepatocyte transplantation.

Shortage of organ donors limits the number of possible liver transplantations. Alternative therapies for treatment of liver failure are currently being developed: (i) extracorporeal artificial liver devices; (ii) bioartificial liver devices using hepatocytes; and (iii) hepatocyte transplantation. The objective of these strategies is to bridge patients with liver failure until a suitable liver allograft is obtained for transplantation or the patient's own liver regenerates sufficiently to resume normal function. In this review, we discuss these strategies and summarize the current status of clinical experience.

Impact of oxygenation of bioartificial liver using perfluorocarbon emulsion perftoran on metabolism of human hepatoma C3A cells.

One of the most important challenges in bioartificial liver designed for patients suffering from acute liver failure is oxygenation of cells within the bioreactor. The aim of this study was to evaluate the impact of oxygenation of bioartificial liver using perfluorocarbon (PFC) emulsion on the metabolic activity of hepatic cells in vitro. Mineral fibers coated with collagen type I were used as scaffolds for hepatic cells. Significantly higher total protein synthesis by hepatic C3A cells cultured in the bioreactor for 24 hours, in the group treated with medium supplemented with PFC emulsion, was observed in comparison with medium without PFC. Albumin production increased in the group treated with PFC after 1 hour of perfusion and was continuously, statistically, significantly higher during perfusion then the control group. In conclusion, the use of oxygen carriers, such as the PFC emulsion, can significantly improve synthetic performance of the bioreactor. Mineral fibers coated with extracellular proteins may serve as support for hepatic cells in the bioreactor.

In vitro analysis of a hepatic device with intrinsic microvascular-based channels.

A novel microfluidics-based bilayer device with a discrete parenchymal chamber modeled upon hepatic organ architecture is described. The microfluidics network was designed using computational models to provide appropriate flow behavior based on physiological data from human microvasculature. Patterned silicon wafer molds were used to generate films with the vascular-based microfluidics network design and parenchymal chamber by soft lithography. The assembled device harbors hepatocytes behind a nanoporous membrane that permits transport of metabolites and small proteins while protecting them from the effects of shear stress. The device can sustain both human hepatoma cells and primary rat hepatocytes by continuous in vitro perfusion of medium, allowing proliferation and maintaining hepatic functions such as serum protein synthesis and metabolism. The design and fabrication processes are scalable, enabling the device concept to serve as both a platform technology for drug discovery and toxicity, and for the continuing development of an improved liver-assist device.

[Therapeutic effect of the latest extracorporal elimination procedure (Prometheus treatment) in acute liver failure caused by intoxication]. / A legújabb extracorporalis eliminációs eljárás (Prometheus-kezelés) terápiás hatékonysága mérgezés okozta akut májelégtelenségben.

INTRODUCTION: Despite intensive therapy the mortality of acute liver failure without organ transplantation is 60-90%. Because of organ shortage in liver transplantation, a significant number of patients dies while being on the waiting list. In order to diminish the mortality, various trials were introduced to remove the albumin-bound and water-soluble toxins in liver failure with the aim to support the spontaneous regeneration of the liver and maintaining the patients alive until liver transplantation. Prometheus treatment is a relatively new technique combining Fractionated Plasma Separation and Adsorption (FPSA) with a high-flux dialysis. During the procedure the patient's own separated albumin-rich plasma passes through special adsorbents making possible the elimination of albumin-bound toxins, while hemodialysis gets rid of water-soluble toxins. AIM: The authors' intention was to demonstrate the efficiency of Prometheus treatment in acute liver failure caused by intoxication. PATIENTS AND METHOD: Prometheus treatment was indicated in three patients who suffered from severe intoxication with paracetamol, potassium permanganate and Amanita phalloides, which resulted in a hepatic failure incurable with conservative therapy. RESULTS: Ten treatments were performed in the three female patients. No serious complication was observed. Due to the treatment the albumin-bound (indirect bilirubin p = 0.048; bile acid p = 0.001) and water-soluble (direct bilirubin p = 0.002; creatinine p = 0.007) toxins were significantly decreased. The level of ammonia, urea nitrogen, fibrinogen and antithrombin III did not change significantly. All the three patients were cured without liver transplantation. CONCLUSION: Prometheus treatment removes efficiently the accumulating toxins in acute liver failure. It is a safe elimination technique. In cases untreatable with conservative therapy it makes possible maintaining the patients alive until the liver regenerates spontaneously, or liver transplantation is feasible.

Clinical experience with molecular adsorbent recirculating system (MARS) in patients with drug-induced liver failure.

The molecular adsorbent recirculating system (MARS) is a novel extracorporeal technique for liver support. We report the clinical results in a group of fourteen patients with drug-induced liver failure. Fourteen patients, aged 22-83 years, with acute or subacute liver failure [mean Child-Turcotte-Pugh (CTP) score 11 (range 8-15)] due to the intake of various drugs (diet pill overdose-2; Chinese traditional medicine (CTM)-4; antibiotic, paracetamol, tuberculostatic, or vasodilator abuse-8) were treated with one to seven sessions of MARS. Beneficial effects such as the improvement of encephalopathy and prothrombin activity, as well as a reduction of bilirubin and ammonia were recorded during MARS treatments. Thirteen out of fourteen patients survived the hospitalization (93%), and two of the discharged patients died during the follow-up of 6-12 months. The overall survival rate was about 79%. MARS therapy can contribute to the improved treatment of drug-induced liver failure patients.

cDNA microarray analysis in hepatocyte differentiation in Huh 7 cells.

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of a hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we previously demonstrated the upregulation of hepatocyte-specific function by spheroid formation in polyurethane foam and by culturing with the histone deacetylase inhibitor, trichostatin A (TSA), in a human hepatoma cell line (Huh 7). In this article we analyze the gene expression profile using cDNA microarray (1281 genes) in spheroid formation or culturing with TSA in Huh 7 to determine the target genes in hepatocyte differentiation. In both the spheroid formation and in the culture with TSA, the Oct-3/4 transcription factor was upregulated more thantwofold, while the early growth response-1 (EGR-1) transactivator was downregulated less than 0.5-fold. These results indicate that expressions of Oct-3/4 and EGR-1 may be key factors in the induction of hepatocyte differentiation in Huh 7.

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells.

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.