Tomoyuki Miyashita

In vivo estimation of bioartificial liver with recombinant HepG2 cells using pigs with ischemic liver failure.

Biological efficacy of a recombinant human hepatic cell line, glutamine synthetase transfected HepG2 (GS-HepG2), was examined with large-scale culture in a circulatory flow bioreactor and in pigs with ischemic liver failure. GS-HepG2 cells were cultured in a circulatory flow bioreactor from 5 × 107 to 4 × 109 cells for 109 days. The cells showed ammonia removal activity even under substrate (glutamic acid)-free medium, suggesting that the GS catalyzed the activity using intracellular glutamic acid that had been pooled during conventional culture. When GS-HepG2 bioartificial liver (BAL) was applied to pigs with ischemic liver failure, survival time was prolonged to 18.8 ± 6.1 h (mean ± SD, n = 4) from 13.8 ± 5.4 h (n = 6) and 10.7 ± 4.1 h (n = 6) (groups treated with cell-free BAL and treated with plasma exchange and continuous hemodia-filtration, respectively). Laboratory data indicated the tendency for improvement in increase of blood ammonia level and decline of blood coagulation indices in the GS-HepG2 BAL-treated group. The advantages and potential for the cell line as a bioreactor in BAL is also discussed, comparing to those of isolated porcine hepatocytes.

Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of a bioartificial liver.

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes. The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 × 107 to 4 × 109 cells. Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient. Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 × 109 GS-HepG2 cells was almost equivalent to 5 × 108 porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-α (TNF-α). Cytokine removal therefore promises to be another useful property of bioreactor cells.

The Significant Improvement of Survival Times and Pathological Parameters by Bioartificial Liver with Recombinant HepG2 in Porcine Liver Failure Model

We developed a bioartificial liver (BAL) containing human hepatoblastoma cell line, HepG2, with the addition of ammonia removal activity by transfecting a glutamine synthetase (GS) gene and estimated the efficacy using pigs with ischemic liver failure. GS-HepG2 cells showed 15% ammonia removal activity of porcine hepatocytes, while unmodified HepG2 had no such activity. The established GS-HepG2 cells were grown in a circulatory flow bioreactor to 3.5–4.1 × 109 cells. Survival time of the animals treated with GS-HepG2 BAL was significantly prolonged compared to the cell-free control (14.52 ± 5.24 h vs. 8.53 ± 2.52 h) and the group treated with the BAL consisting of unmodified wild-type HepG2 (9.58 ± 4.52 h). Comparison showed the cell-containing BAL groups to have significantly fewer incidences of increased brain pressure. Thus, the GS-HepG2 BAL treatment resulted in a significant improvement of survival time and pathological parameters in pigs with ischemic liver failure.

Standardization of Ischemic Hepatic Failure in Pigs as a Model for Bioartificial Liver Assessment

AbstractPurpose. A standard protocol of ischemic liver failure in pigs was examined to establish a system for assessing the efficacy of a bioartificial liver, based on clinical practice. Methods. The portal blood flow was extracorporeally bypassed into the cervical jugular vein, using a centrifugal blood pump. The portal vein and hepatic artery were then ligated. Results. The maintenance protocol was established as follows: (1) the concentration of the inhaled anesthetic was decreased by 0.2% when the systolic blood pressure was ≪100 mmHg; (2) the volume of an infusion containing 5% glucose was increased to 10 ml/kg per hour when central venous pressure was ≪5 mmHg; (3) 20 ml of 50% glucose was injected intravenously when the blood glucose was ≪50 mg/dl; (4) 2000 units of heparin was injected intravenously when the activated clotting time was ≪150 s; (5) sodium bicarbonate was given when the blood pH was ≪7.3; (6) tidal volume was increased by 1 ml/kg when the pCO2 was ≫80 mmHg; (7) oxygen was increased by 25% when the pO2 was ≪100 mmHg. No vasopressors were used in the experiment. Conclusion. Our protocol reduced the operating time and minimized the risk of data deviation that can arise from variations in operating techniques and individual animal conditions. This experimental model is also easy to use as a bridge to transplantation.

Application of circulatory flow bioreactor for long-term and large-scale culture of glutamine synthetase transduced CHO cells and its ammonia removal activity with an aim of development for a bioartificial liver assist system

Glutamine synthetase-transduced Chinese hamster ovary (GS-CHO) cells were cultured in a circulatory flow bioreactor for 28 days. The bioreactor inoculated with 4.72×108 of GS-CHO cells was kept at 37°C and supplied with air, CO2, O2, and glutamine-free RDF culture medium containing 5% fetal bovine serum, 2.3mM of ammonia, and 1.0mM of glutamate. Since GS-CHO cells synthesize glytamine from ammonia and glutamate, the ammonia concentration in the culture medium was decreased corresponding to the cell growth. The clearance rate of added ammonia showed that the ammonia removal activity was equivalent to that of 1.25×108 porcine hepatocytes. Amino acid analysis showed a peak of glutamine synthesis that was not found in the original medium. The ammonia concentration in the culture medium returned to the original level 24 days after the start of culture, suggesting the cells went into a stationary stage. The number of cells in the bioreactor at the end of the culture was 9.64×109, and the cells were uniformly distributed throughout the glass fiber cell matrix. These data suggest that the circulatory flow bioreactor system with recombinant cells is applicable for a bioartificial liver assist unit to remove toxic substances in the blood of a patient whose detoxification has been injured by hepatic failure.

Effect of ethanolamine on liver regeneration after 90% hepatectomy in rats.

SURGICAL PROCEDURES, such as extended liver resection or living-related liver transplantation may be performed because normal liver has vigorous regenerative potential. However, reduced hepatic volume can produce postoperative hepatic insufficiency, which is a serious problem. After extensive liver resection for hepatic tumor or in living-related liver donors maximum volume of liver resection or thus the minimum volume of donor liver are limited. When the volume exceeds these limits, surgical treatment is not indicated. However, a new treatment that promotes postoperative liver regeneration would extend these limits. Two approaches have been attempted to promote liver regeneration. One approach prepares the host environment for liver regeneration by plasma exchange. In recent years, favorable results also have been reported using a bioartificial liver as a bridge. The other approach is promotion of liver regeneration using hepatocyte growth factor (HGF) or ethanolamine (EA). EA is a nutrient factor derived from the small intestine. In this study, we investigated the enhancing effect of EA using a 90% partial hepatectomy (PHX) rat model.

A bioreactor with glutamine synthetase-transfected recombinant HepG2 cells exhibits ammonia removal activity without the need for added cofactors and substrates: Advantage of a cellular bioreactor over enzyme-immobilized beads

The biological efficacy of recombinant human hepatic cell line, glutamine synthetase-transfected HepG2 (GS-HepG2), was examined under various culture conditions in large-scale culture. GS-HepG2 (2×109 cells) were cultured in a circulatory flow bioreactor, and the changes in the concentrations of ammonia, glucose, glutamine, and glutamic acid were checked with normal, glutamic acid-insufficient, and glutamine-excess medium or porcine plasma. Whereas the glutamine synthetase reaction required high concentrations of Mg2+ in vitro, GS-HepG2 removed ammonia in the culture medium without the addition of Mg2+. Similarly, ammonia removal activity was observed in the medium with physiological concentrations of glutamic acid far lower than the enzymeKm. Moreover, product-induced inhibition of the enzyme activity was not observed with addition of excess glutamine to the medium. Ammonia removal activity was also detected in porcine plasma at the level equivalent to that in the culture medium. The substrates and cofactors indispensable to the glutamine synthetase reaction were likely to be generated and/or stored inside the cells. Thus, the bioreactor with recombinant cells catalyzes the reaction without the need for added cofactors and substrates and surpasses the enzyme-immobilized system to perform blood dialysis by selective removal of toxic substances.