• KSBB Journal
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• v.29 no.1
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• pp.58-66
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• 2014

Demand for in vitro pharmacological evaluation and toxicity test using human hepatocytes has been increasing. In USA and Europe, human hepatocytes obtained from donated whole liver unsuitable for transplantation were distributed to researchers and deposited in cell bank facility as cryopreserved vial. In Korea, however, incidence of transplantation- inappropriate whole liver has been quite low and the whole livers almost have so severe liver disease such as fatty or fibrotic liver that cannot meet the demand. In this study we aimed to isolate human hepatocytes from liver resection surgery-originated partial liver, and assure the isolated human hepatocytes and its cryopreserved hepatocytes to be qualified for the in vitro pharmacological evaluation and drug toxicity tests. We compared those with commercially available human hepatocyte, BD $GenTest^{TM}$ by cell morphology, hepatic gene expression, urea synthesis, albumin secretion, ammonia removal, and cytochrome P450 induction activities. Changes in hepatotoxic gene expression after cryopreservation are evaluated with a typical hepatotoxic drug, acetaminophen. Consequently, the fresh hepatocytes from the partial liver and its cryopreserved hepatocytes expressed their intrinsic hepatic functions well and showed equal hepatotoxicity gene expression trend regardless to cryopreservation. Therefore, liver resection surgery-originated partial liver can be used as a useful source of human hepatocytes for various pharmacological and hepatotoxicity test.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.305-306
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• 2003

To treat fulminant hepatic failure (FHF) patients, various extracorporeal bioartificial liver (BAL) systems have been developed. Several requirements should be met for the development of BAL systems: hepatocytes should be cultured in a sufficiently high density; their metabolic functions should be of a sufficiently high level and duration; and the BAL systems module should permit scaling-up and aseptic handling. Several investigators have found that freshly isolated primary hepatocytes can be cultured into three dimensional, tightly packed, freely suspended, multicellular aggregates, or spheroids. These specialized cell structures exhibited enhanced liver specific functions and a prolonged differentiated state compared to cells maintained in a monolayer culture. Cells in spheroids appear to mimic the morphology and ultrastructure of the in vivo liver lobule. The ability of hepatocytes to organize into three-dimensional structures was hypothesized to contribute to their enhanced liver-specific activities. In this study, the ammonia removal rate and urea secretion rate of pig hepatocytes spheroids encapsulated in Ca-alginate bead were determined. A packed-bed bioreactor with encapsulated pig hepatocytes was devised as BAL support system. The efficacy of the system was evaluated in vitro.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.5
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• pp.617-624
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• 2013

The purpose of this study is to investigate the protective effects of 25 herbal formulas on acetaminophen (APAP) or D-galactosamine (D-GalN)-induced hepatotoxicity in primary rat hepatocytes. Cell viability was measured using by Cell Counting Kit-8. 15 kinds of herbal formulas significantly reversed the cell viabilities of D-GalN-treated rat hepatocytes compared with D-GalN alone (p<0.05). In particular, 9 herbal formulas (Bangpungtongseong-san, Bojungikgi-tang, Galgeun-tang, Gumiganghwal-tang, Guibi-tang, Sagunja-tang, Samsoeum, Pyeongwi-san and Yijin-tang) showed the potent protective effects. However, 8 herbal formula exerted weak protective effects and 2 herbal formula did not exert effects on hepatotoxicity by D-GalN. On APAP-induced hepatotoxicity, 7 kinds of herbal formulas increased the viabilities of hepatocytes compare with APAP alone (p<0.05). These results could be provide a valuable information for the future in vivo or clinical studies to predict the hepatoprotective effects of herbal formulas.

• Macromolecular Research
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• v.11 no.6
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• pp.488-494
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• 2003

We have fabricated gelatin-immobilized polyurethane foams (PUFG) by dipping polyurethane foam (PUF) in an aqueous solution containing gelatin and by subsequent reaction with glutaraldehyde after freeze-drying. Gelatin aqueous solutions of different concentrations were used as the dipping solutions to control the amount of immobilized gelatin. The average pore size of PUF decreased with an increase in gelatin concentration. It was found from the hepatocyte adhesion experiment that the amount of hepatocytes seeded on PUFG1, prepared by using a 1% aqueous gelatin solution, was higher than that on other PUFGs. The hepatocytes inoculated in PUFG1, were slightly aggregated as the incubation time increased. The cells inoculated in PUFG1 showed higher ammonia removal ability than those monolayer-cultured on a gelatin-immobilized polystyrene dish (PSG) after 1 and 4 days of incubation time. The inoculated cells exhibited higher albumin secretion relative to monolayer-cultured hepatocytes on PSG. Albumin secretion by hepatocytes seeded on PUFG1 was increased by the presence of serum and was further increased by both the presence of serum and cytokines. The results obtained from a 3-(3,4-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that PUFG can provide a better microenvironment for hepatocyte culture along with nutrition and metabolite transfer through the high porosity of PUF.

• Toxicological Research
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• v.22 no.3
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• pp.301-306
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• 2006

Microcystin-LR(MC-LR), a cyanobacterial toxin produced by Microcystis aeruginosa, causes severe hepatotoxicity. Here we investigated the morphologic changes of rat hepatocyte spheroid induced by exposure of MC-LR($10^{-6}M$) in vitro. In addition, to determine the effects of such toxin in the process of hepatocyte spheroid formation, primarily isolated hepatocytes were incubated with MC-LR and the process of spheroid formation was observed. In both hepatocyte spheroid and suspension culture systems, the morphologic changes caused by MC-LR were noticible at 5 min post exposure and were characterized by the loss of microvilli, cytoplasmic vacuolation, the accumulation of lipid droplets, and blob formation. Especially, the size and numbers of blob on the cell surface were increased as the incubation time prolonged and the appearance of electron dense bodies were observed in the cytoplasm of hepatocyte at 20 min post exposure. Furthermore, bile canaliculi-like structures in the hepatocyte spheroids were slightly widened and the process of spheroids formation was inhibited in the isolated hepatocytes incubated with MC-LR. These results indicate that morphologic changes in. the hepatocyte membrane and organelles seem to be typical events in showing the MC-LR induced hepatotoxic effects and the spheroid culture method might be a useful experimental tool to evaluate hepatoxicity since it reflects the in vivo status of hepatocytes.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.3
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• pp.190-195
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• 2007

In the present study, the author investigated to the cytotoxocity in cultured rat hepatocytes of Viscum album lectin. The cytotoxcity effect in Viscum album lectin on the activity of LDH was also investigated. Viscum album lectin significantly increased LDH leakage into medium of hepatocytes treated or untreated with $CCl_4$ (p<0.001). However, Viscum album lectin significantly increased LDH leakage from $CCl_4$-induced hepatocyte (p<0.001). There was a significant increase in LDH levels relative to the control group. Histological observation basically supported the result obtained from LDH assay. The livers of rats challenged with $CCl_4$ produced a marked increased cytoplasmic vacuoles and inflammatory cells in number, while the number of necrotic cells and swollen hepatocytes did not change significnatly. Rats administered DMSO alone did not alter the normal hepatic architecture. Histological observation of liver section in rat treated 72 hrs with either Viscum album lectin $CCl_4$-induced liver damage showed number of cytoplasmic vaculoe and necrotic cell. The number of inflammatory cell increased markedly. This results suggest to the conclusion that Viscum album lectin has a effect of hepatotoxicity activator.

• IMMUNE NETWORK
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• v.10 no.4
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• pp.126-134
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• 2010

Background: $CD8^+$ T cells contribute to the clearance of Hepatitis B virus (HBV) infection and an insufficient $CD8^+$ T cell response may be one of the major factors leading to chronic HBV infection. Since the HBx antigen of HBV can up-regulate cellular expression of several immunomodulatory molecules, we hypothesized that HBx expression in hepatocytes might affect $CD8^+$ T cell activity. Methods: We analyzed the activation and apoptosis of $CD8^+$ T cells co-cultured with primary hepatocytes rendered capable of expressing HBx by recombinant baculovirus infection. Results: Expression of HBx in hepatocytes induced low production of $interferon-{\gamma}$ and apoptosis of CD8+ T cells, with no effect on CD8 T cell proliferation. However, transcriptional levels of H-2K, ICAM-1 and PD-1 ligand did not correlate with HBx expression in hepatocytes. Conclusion: Our results suggest that HBx may inhibit $CD8^+$ T cell response by regulation of $interferon-{\gamma}$ production and apoptosis.

• Toxicological Research
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• v.19 no.1
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• pp.67-72
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• 2003

Cholestatic liver injury results from the accumulation of toxic bile salts within the liver. The aim of the present study is to elucidate the changes in expression and cellular localization of apoptosis related proteins in the liver of bile duct-ligated (BDL) rat. Extrahepatic cholestasis was induced by double ligation of the common bile duct and cut between the ligatures. Animals were sacrificed at day 3 and at week 1, 2, 4, 6, and 8 after BDL. The number of TUNEL positive cells was increased significantly after 3 days of BDL, decreased over 2 weeks and remained constant thereafter. Fas expression was not changed and activation of caspase 8 did not occur. Fas immunoreactivity was exclusively observed in the cytoplasm of hepatocytes, indicating that Fas expressed in rat hepatocytes is a soluble form. Hepatocyte apoptosis was associated with Bax expression, which showed a peak at day 3 and decreased over time gradually. Immnunostaining of Bax was observed in hepatocytes and bile duct epithelial cells (BEC) of control and BDL rats. Bcl-2 was increased over time in BDL rats. These results suggest that apoptosis of hepatocytes in BDL rats is independent of Fas and controlled by Bax expression.

• YAKHAK HOEJI
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• v.36 no.1
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• pp.80-86
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• 1992

Streptozotocin (STZ) is a naturally occurring nitrosoamide used extensively to produce diabetes in experimental animals. Our previous study has demonstrated that i.v. administraton of streptozotocin induces significant red blood cell hemolmysis in rats. Since it has been reported that the highest concentration of STZ is found in the liver, the effect of STZ in freshly isolated rat hepatocytes has been investigated. STZ treatment (10 mM) did not cause significant loss of viability throughout 4 hour incubation, while high dose of STZ (300 mM) to hepatocytes resulted in complete cell death within 3 hours. Addition of 40 mM glucose to incubation medium did not potentiate STZ-induced hepatotoxicity, suggesting that STZ-induced hyperglycemia in vivo did not affect its hepatotoxicity. To investigate the mechanixm of the toxicity, intracellular total glutathione level was determined. Tratment with 10 mM STZ which was not toxic to hepatocytes led to complete depletion of intracellular glutathione level within 1 hour incubation. These results suggest that STZ-induced hepatotoxicity may be independent on the intracellular glutathione depletion.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.11a
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• pp.135-140
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• 1994

Over past decades, numerous in vitro model has been developed to investigate drug metabolism. In the order of complexity we found the isolated perfused liver, hepatocytes in co-culture with epithelial cells, hepatocytes in suspension and in primary culture and subcellular hepatic microsomal fractions. Because they can be easily prepared from both animals (pharmacological and toxicological species) and humans (whole livers as well as biopsies obtained during surgery) hepatocytes in primary culture provide the most powerful model to better elucidate drug behavior at an early stage of preclinical development such as : 1. the characterization of main biotransformation reactions. 2. the identification of phase I and phase II isozymes involved in such reactions 3. the evaluation of interspecies differences allowing the selection of a second toxicological animal species more closely related to man on the basis of metabolic profiles 4. the detection of the inducing and/or inhibitory effects of a drug on metabolic enzymes, the prediction of drug interactions 5. the estimation of inter-individual variability in biotransformation reactions. The use of hepatocytes, and in particular those obstained from humans, at an early stage of drug development allows the obtention of more predictive preclinical data and a better knowledge of drug behavior in humans before the first administration of the drug in healthy volunteers.