"Off-The-Shelf" Bioartificial Liver Support System Using Cryopreserved Immobilized Hepatocyte Spheroids in a Porcine Acute Liver Failure Model.

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.

The aim of this study was to evaluate a novel bioartificial system in a canine model of acute liver failure. An acute liver failure model in canines was induced by an end-side portocaval shunt combined with common bile duct ligation and transection. The bioartificial liver system, which utilized blood perfusion through a hollow fiber bioreactor from BIOLIV A3A inoculated with 1.0 - 3.1 x 1010 porcine hepatocyte spheroids, was developed for the treatment of acute liver failure. Sixteen acute liver failure model canines were divided between a group treated with bioartificial liver (n=8) and a control group (n=8) for 5 h. Blood alanine aminotransferase (ALT), alkaline phosphatase (AKP), total bilirubin (TBi), direct bilirubin (DBi), prothrombin time (PT), ammonia levels, and the ratio of branched chain to aromatic amino acids (Fischer's ratio) were determined. ALT, AKP, TBi, DBi, and ammonia levels were significantly elevated, PT was significantly prolonged, and Fischer's ratio decreased significantly in the canine model of the two groups on day 14 after operation compared to baseline. There were no significant differences between the two groups in laboratory data before treatment. In canines treated with the bioartificial liver system, ALT, AKP, TBi, DBi, and ammonia levels decreased significantly, PT was significantly shortened, Fischer's ratio was significantly elevated after treatment, and the survival rate by day 7 after treatment was 100%. In canines in the control group, on the other hand, there were no significant differences in ALT, AKP, TBi, DBi, PT, and ammonia levels between pretreatment and posttreatment, though these indices decreased to a slight degree after treatment. The survival rate by day 7 after treatment was 62.5% in the control group. Fischer's ratio decreased after treatment. ALT, AKP, TBi, DBi, PT, and ammonia levels in the bioartificial liver system group were lower, and Fischer's ratio and survival rate were higher than those in the control group after treatment. These results indicate that the novel bioartificial liver system we developed has a significant impact on the course of canine acute liver failure model and has potential advantages for clinical use in patients with acute liver failure.

To evaluate the functions of a new bioartificial liver (BAL) system in vitro and in vitro. The BAL system was configured by inoculating porcine hepatocyte spheroids into the cell circuit of a hollow fiber bioreactor. In the experiments of BAL in vitro, the levels of alanine aminotransferase (ALT), total bilirubin (TB), and albumin (ALB) in the circulating hepatocyte suspension and RPMI-1640 medium were determined during 6 h of circulation in the BAL device. In the experiments of BAL in vitro, acute liver failure (ALF) model in canine was induced by an end-side portocaval shunt combined with common bile duct ligation and transaction. Blood ALT, TB and ammonia levels of ALF in canines were determined before and after BAL treatment. During 6 h of circulation in vitro, there was no significant change of ALT, whereas the TB and ALB levels gradually increased with time both in the hepatocyte suspension and in RPMI-1640 medium. In the BAL treatment group, blood ALT, TB and ammonia levels of ALF in canines decreased significantly. The new BAL system has the ability to perform liver functions and can be used to treat ALF.

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.

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.

Potentiality of Immobilized Pig Hepatocyte Spheroids in Bioartificial Liver System

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.

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.

Both the large variety of liver functions for maintaining body homeostasis and the proven effectivity of whole liver transplantation in the therapy of acute liver failure (ALF), are important reasons to presume that cell-free liver support systems will not be able to adequately support the failing liver. Accordingly, bioartificial liver (BAL) systems have shown their efficacy in experimental ALF models in small and large animals, and have shown to be suitable and safe in phase 1 studies in humans with ALF. However, the optimal BAL system is still under development. Important issues are the source of the cellular component and the configuration of the BAL system with regard to cell attachment, mass transfer characteristics and oxygenation at site. The deficiency of all BAL systems to excrete bile effectively is another important topic for improvement. The great challenge for the future is to develop a well-functioning and safe human hepatic cell line which can replace the widely used porcine (xenogeneic) hepatocytes. Theoretically, a combination of a cell-free liver support system and a BAL system might be optimal for the treatment of ALF patients in the near future.

AIMTo establish a reversible porcine model of acute liver failure (ALF) and treat it with an artificial liver system.METHODSSixteen pigs weighing 30-35 kg were chosen and administered with acetaminophen (APAP) to induce ALF. ALF pigs were then randomly assigned to either an experimental group (n = 11), in which a treatment procedure was performed, or a control group (n = 5). Treatment was started 20 h after APAP administration and continued for 8 h. Clinical manifestations of all animals, including liver and kidney functions, serum biochemical parameters and survival times were analyzed.RESULTSTwenty hours after APAP administration, the levels of serum aspartate aminotransferase, total bilirubin, creatinine and ammonia were significantly increased, while albumin levels were decreased (P < 0.05). Prothrombin time was found to be extended with progression of ALF. After continuous treatment for 8 h (at 28 h), aspartate aminotransferase, total bilirubin, creatinine, and ammonia showed a decrease in comparison with the control group (P < 0.05). A cross-section of livers revealed signs of vacuolar degeneration, nuclear fragmentation and dissolution. Concerning survival, porcine models in the treatment group survived for longer times with artificial liver system treatment (P < 0.05).CONCLUSIONThis model is reproducible and allows for quantitative evaluation of new liver systems, such as a bioartificial liver. The artificial liver system (ZHJ-3) is safe and effective for the APAP-induced porcine ALF model.

To establish a highly reproducible animal model of acute liver failure (ALF), for assessing the effect of bioartificial liver support system (BALSS). A two-phase complete liver devascularization procedure was performed in eight loco-hybrid pigs. Blood biochemical index and liver biopsy were studied every 2 h after surgery, and survival time was recorded. The BALSS constructed with high volume recirculating technique was a hollow fiber circulating system consisting of a hepatocyte reactor-hollow fiber module inoculated with microcarrier-adhering hepatocytes, and a double pump, heparinized, thermostabilized, micro-capsulized activated carbon-adsorbing plasmapheresis system. Twelve pigs undergoing two-phase surgery were randomized into: control group (perfused without hepatocytes, n = 6) and treatment group (perfused with hepatocytes, n = 6). Intergroup liver biochemical indexes, survival time, and liver pathological changes were analyzed at regular intervals. Two-phase surgery was performed in all the experimental pigs, and there was no obvious difference between their biochemical indexes. After 3 h of phase II surgery, ammonia (Amm) increased to (269+/-37) micromol/L. After 5 h of the surgery, fibrinogen (Fib) decreased to (1.5+/-0.2) g/L. After 7 h of the surgery, ALT, AST, Tbil and PT were (7.6+/-1.8) nka/L, (40+/-5) nka/L, (55+/-8) micromol/L and (17.5+/-1.7) nka/L respectively. After 9 h of surgery, ALB and Cr were (27+/-4) g/L and (87+/-9) micromol/L. After 13 h of surgery, BUN was (3.5+/-0.9) micromol/L. All the above values were different from those determined before surgery. Survival time of pigs averaged 13.5+/-1.4 h. ALF pigs in the other group were treated with BALSS. The comparison analysis between the treated and control animals showed the changes of Tbil, PT, Alb, BUN, Cr, Fib, and Amm (P<0.01), but there was no change of ALT and AST. The survival time was statistically different (P<0.01), and there was no significant difference in histological changes. The porcine ALF model established by two-phase devascularized surgery is valid and reproducible. The hollow fiber BALSS can meet the needs of life support and is effective in treating ALF.

Objective To investigate the influence of membrane molecular weight cut off in our bioartificial liver(BAL)system.Methods Beagle dogs were used for a model of acute liver failure through D-galactosamine administration.The acute liver failure Beagles were divided into two groups by the membrane molecular weight cut off.Group A was treated with BAL containing 200 kDa retention rating membrane.Group B was treated with BAL containing 1200 kDa retention rating membrane.Each group underwent two six-hour BAL treatments that were performed on day 1 and day 21.BAL medium were examined and levels of IgG,IgM,and complement hemolytic unit of 50％(CH50)antibodies were measured in all Beagles and.Results BAL treatment was associated with a significant decline in levels of CH50.1200 kDa group experienced a significant increase in levels of IgG and IgM after two BAL treatments.Significant levels of canine proteins were detected in BAL medium from 1200 kDa group.Conclusions Xenogeneic immune response in the BAL system was influenced by membrane molecular weight cut off. Key words: Bioartificial liver; Membrane molecular weight cut off; Porcine hepatocyte; Bone marrow mesenchymal stem cells

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.

Extracorporeal liver support (ELS) may play a role in bridging therapy in patients with acute liver failure (ALF). The aim of this study was to compare the influence of nonbiological and biological methods on intracranial pressure (ICP) in an animal model of ALF. A surgical devascularization model of ALF in pigs (35-40 kg) was used. Elimination therapy started after the onset of hypoglycemia. Biochemical parameters (bilirubin, ammonia, lactate, etc.) as well as ICP and cerebral perfusion pressure (CPP) were monitored for 12 hours. Of the total 31 pigs with ALF, 14 animals were treated by fractionated plasma separation and absorption (FPSA), 10 were treated with a bioartificial liver (BAL), and 7 animals were used as a control group. FPSA and BAL treatment started on average 3 hours 17 minutes and 2 hours 21 minutes, after devascularization and lasted for 5 hours 54 minutes and 5 hours 43 minutes, respectively. Ammonia levels were lower in the FPSA group, and bilirubin levels differed significantly in both the FPSA and BAL groups compared with controls. However, ICP values were reduced more effectively in pigs treated by FPSA: 19.1 vs. 27.0 mm Hg at 9 hours, 22.5 vs. 28.7 mm Hg at 11 hours, and 24.0 vs. 33.0 mm Hg at 12 hours (p<0.05). The artificial liver support system FPSA reduced ICP values more effectively than the Performer O. Liver RanD BAL system. Compared with this BAL system, the nonbiological elimination method of FPSA is a simpler application with the advantage that it can be applied in a more continuous way.

We have developed a bioartificial liver support system utilizing hollow-fiber bioreactor, plasmapheresis and microcarrier cell culture technologies. Liver cells were obtained through portal vein perfusion with ethylenediaminetetraacetate or ethylenediaminetetraacetate/collagenase. A mathematical model of mass transport in a hollow-fiber module, at various plasma flow velocities and system configurations, was developed. The bioartificial liver's ability to carry out specific differentiated metabolic liver functions was tested in vitro and in vivo. A reproducible large-animal model of acute ischemic liver failure was developed. Most major first-generation cyclosporine and 19-norterstosterone metabolites were isolated after substrate addition to the bioartificial liver in vitro. After bioartificial liver treatment for 6 hr (with dog or pig liver cells), dogs with acute liver failure had significantly lower serum ammonia and lactate levels and significantly higher serum glucose levels than did control animals treated with a bioartificial liver system inoculated with microcarriers alone. In addition, bioartificial liver–treated animals had significantly higher mean systolic blood pressures than did controls. Liver cell viability at the end of the 6-hr in vivo experiment was greater than 90%. (HEPATOLOGY 1993;17:258–265.)