Thomas Schreiter

Cytochrome P450-Dependent Metabolism in HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor

Reliable and stable in vitro cellular systems maintaining specific liver functions important for drug metabolism and disposition are urgently needed in preclinical drug discovery and development research. The cell line HepaRG exhibits promising properties such as expression and function of drug-metabolizing enzymes and transporter proteins, which resemble those found in freshly isolated human hepatocytes. In this study, HepaRG cells were cultured up to 68 days in a three-dimensional multicompartment capillary membrane bioreactor, which enables high-density cell culture under dynamic conditions. The activity of drug-metabolizing cytochrome P450 (P450) enzymes was investigated by a cocktail of substrates for CYP1A1/2 (phenacetin), CYP2C9 (diclofenac), CYP2B6 (bupropion), and CYP3A4 (midazolam). The model P450 substrates, which were introduced to the bioreactor system mimicking in vivo bolus doses, showed stable metabolism over the entire experimental period of several weeks with the exception of bupropion hydroxylase, which increased over time. Ketoconazole treatment decreased the CYP3A4 activity by 69%, and rifampicin induced the CYP3A4- and CYP2B6-dependent activity 6-fold, which predicts well the magnitude of changes observed in vivo. Moreover, polarity of transporter expression and formation of tissue-like structures including bile canaliculi were demonstrated by immune histochemistry. The long-lasting bioreactor system using HepaRG cells thus provides a promising and stable liver-like in vitro model for continuous investigations of the hepatic kinetics of drugs and of drug-drug interactions, which well predict the situation in vivo in humans.

Preservation of the Synthetic and Metabolic Capacity of Isolated Human Hepatocytes by Coculture With Human Biliary Epithelial Cells

Bioartificial liver support systems have demonstrated limited efficacy in compensation of liver detoxification and substitution of liver‐derived factors. However, in these devices, the biological substitution of the complex liver function has been restricted to xenogeneic or transformed hepatocytes. Therefore, we have examined the long‐term effect of coculturing normal human hepatocytes (HCs) with allogeneic biliary epithelial cells (BECs). We applied functional in vitro assays to examine their metabolic potential by ammonia detoxification to urea, cytochrome P450‐dependent lignocaine conversion to mono‐ethyl‐glycine‐xylidide (MEGX), and protein expression and secretion. As the liver has a pivotal role in the synthesis of coagulation factors, we measured antithrombin III (AT III), factor VII, and albumin, comparing HCs plated on collagen or inside 3‐dimensional collagen gels. Over 30 days, expression and secretion of albumin and clotting factors by human HCs were augmented by culture inside collagen gel, but were also enhanced and better maintained by coculture with BECs. Higher proportions of BECs cocultured with HCs substantially increased the protein synthesis and urea production. Remarkably, the almost absent cytochrome P450 activity of HC alone after 1 week could be reversed and maintained over 3 weeks by coculture with BECs. The pattern of these effects differed from the extent of interleukin‐6 (IL‐6) production and HC viability under the compared conditions. In conclusion, coculture of human HCs with BECs impressively restores the synthetic and metabolic liver function in vitro. These results suggest mechanisms of improved liver epithelial differentiation supported by coculture conditions. This technique offers new perspectives in bioartificial liver support, hepatocyte transplantation, and stem cell differentiation. (Liver Transpl 2005;11:410–419.)

Asialoglycoprotein receptor facilitates hemolysis in patients with alcoholic liver cirrhosis

Hemolysis in patients with advanced alcoholic liver disease is a common clinical problem and indicates an unfavorable prognosis. In many cases, the etiology of the hemolysis remains unknown. We observed three patients with alcoholic liver disease, suffering from severe hemolytic anemia, requiring multiple blood transfusions. Steroid therapy was ineffective and two of the patients died. All patients had a soluble variant of the human asialoglycoprotein receptor (s‐ASGP‐R) in their serum, as well as high titers of autoantibodies against this receptor (anti‐ASGP‐R). Consecutively, examination of 60 patients with alcoholic liver disease revealed a high incidence for s‐ASGP‐R (36%) and anti‐ASGP‐R (27%) in patients with alcoholic liver cirrhosis (ALC) compared to patients with cirrhosis due to viral hepatitis. The potential etiology of hemolysis was studied in vitro on erythrocytes from patients with ALC and from healthy donors. Isolated ASGP‐R but not anti‐ASGP‐R bound to the surface of erythrocytes preferentially of blood group A1 and caused dose‐dependent agglutination and hemolysis, while this phenomenon was much lower using erythrocytes of the blood group B and almost absent with blood group O‐erythrocytes. Furthermore, agglutination and hemolysis only occurred in erythrocytes from ALC‐patients or after the pre‐treatment of cells with neuraminidase. ASGP‐R induced agglutination and hemolysis was blocked by the competitive ASGP‐R inhibitor asialofetuin. In conclusion, our results indicate a new, non‐immunological mechanism for hemolysis in patients with alcoholic liver disease, mediated through agglutination by a soluble variant of the human asialoglycoprotein receptor and mechanical shear stress. (HEPATOLOGY 2004;39:1398–1407.)

Human Ex-Vivo Liver Model for Acetaminophen-induced Liver Damage.

Reliable test systems to identify hepatotoxicity are essential to predict unexpected drug-related liver injury. Here we present a human ex-vivo liver model to investigate acetaminophen-induced liver injury. Human liver tissue was perfused over a 30 hour period with hourly sampling from the perfusate for measurement of general metabolism and clinical parameters. Liver function was assessed by clearance of indocyanine green (ICG) at 4, 20 and 28 hours. Six pieces of untreated human liver specimen maintained stable liver function over the entire perfusion period. Three liver sections incubated with low-dose acetaminophen revealed strong damage, with ICG half-lives significantly higher than in non-treated livers. In addition, the release of microRNA-122 was significantly higher in acetaminophen-treated than in non-treated livers. Thus, this model allows for investigation of hepatotoxicity in human liver tissue upon applying drug concentrations relevant in patients.

168 NOVEL EX-VIVO PERFUSION SYSTEM: FUNCTIONAL CHARACTERISTICS OF HUMAN NON-CIRRHOTIC AND CIRRHOTIC LIVER SECTIONS MIRROR PATIENTS’ CLINICAL PARAMETERS

168 NOVEL EX-VIVO PERFUSION SYSTEM: FUNCTIONAL CHARACTERISTICS OF HUMAN NON-CIRRHOTIC AND CIRRHOTIC LIVER SECTIONS MIRROR PATIENTS’ CLINICAL PARAMETERS T. Schreiter, G. Marquitan, M. Darnell, J.-P. Sowa, M. BrockerPreus, T.B. Andersson, H.A. Baba, M. Furch, U. Rauen, Z. Mathe, J. Treckmann, G. Gerken, R.K. Gieseler, A. Canbay. Clinic for Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany; DMPK Innovative Medicines, AstraZeneca RD Dept. of Endocrinology, Inst. for Pathology and Neuropathology, University Hospital Essen, Essen, Rodos BioTarget GmbH, Hannover, Inst. for Physiological Chemistry, Clinic for General, Visceral and Transplantation Surgery, University Hospital Essen, Essen, Germany E-mail: ali.canbay@uni-due.de