Use Of Human Hepatocytes Research Articles

Expression and functional activity of cytochrome P450 enzymes in human hepatocytes with sustainable reproducibility for in vitro phenotyping studies.

Primary human hepatocytes are an essential in vitro tool for evaluating drug metabolism, drug-drug interactions, and hepatotoxicity. This model is considered as the gold standard in matter of DMPK studies in both industrial and academic research. The primary human hepatocytes are used either in suspension or in monolayer, as fresh or frozen cells. However, the use of this model is limited due to the lack of availability, rapid loss of functionality, high cost as well as the variable hepatocyte plating efficiencies in culture and the limited stock of hepatocytes derived from the same origin. Chimeric TK-NOG mice with humanized livers (humanized liver mice) are an attractive platform for drug metabolism and toxicity, which were produced by transplanting human hepatocytes into immunodeficient mice with injured livers. Here, we show that, using humanized mouse liver, in vivo human hepatocyte repopulation was over ~100-fold enabling the continuous and abundant use of human hepatocytes of the same origin and improving their plateability. In our latest cell preparations, hepatocytes isolated from humanized liver mice (Hu-Liver cells) exhibited high purity (ratio of HLA-positive cells: 92±3%), good viability (75±12%), and yield (1.0×108 cells/mouse). Human hepatic drug metabolizing enzymes, transporters, and nuclear receptors genes were expressed in humanized mouse liver. Drug-metabolizing activities in Hu-Liver cells were comparable to or higher than those in primary human hepatocytes. An extensive P450-dependent human drug metabolism was observed in Hu-Liver cells. CYP1A2, CYP2B6, and CYP3A4/5 activities/mRNA in Hu-Liver cells were induced by the hepatocyte exposure to typical human P450 inducers, omeprazole, phenobarbital, and rifampicin, respectively. Finally, Human albumin secretion and CYP3A-mediated drug oxidation activity were maintained over 4-weeks. Altogether, the expression level of pharmacokinetics-related genes, enzyme activity, human-typed drug metabolism, and inducibility of P450 in Hu-Liver cells make from humanized mouse liver a relevant and robust model for in vitro preclinical studies, including drug metabolism, pharmacokinetics, and toxicology studies.

Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells.

Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid 2-related factor 2 (Nrf2)-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

Evaluation of Adverse Drug Properties with Cryopreserved Human Hepatocytes and the Integrated Discrete Multiple Organ Co-culture (IdMOC(TM)) System.

Human hepatocytes, with complete hepatic metabolizing enzymes, transporters and cofactors, represent the gold standard for in vitro evaluation of drug metabolism, drug-drug interactions, and hepatotoxicity. Successful cryopreservation of human hepatocytes enables this experimental system to be used routinely. The use of human hepatocytes to evaluate two major adverse drug properties: drug-drug interactions and hepatotoxicity, are summarized in this review. The application of human hepatocytes in metabolism-based drug-drug interaction includes metabolite profiling, pathway identification, P450 inhibition, P450 induction, and uptake and efflux transporter inhibition. The application of human hepatocytes in toxicity evaluation includes in vitro hepatotoxicity and metabolism-based drug toxicity determination. A novel system, the Integrated Discrete Multiple Organ Co-culture (IdMOC) which allows the evaluation of nonhepatic toxicity in the presence of hepatic metabolism, is described.

The history and use of human hepatocytes for the treatment of liver diseases: the first 100 patients.

Orthotopic liver transplantation remains the only curative treatment for many end-stage liver diseases, yet the number of patients receiving liver transplants remains limited by the number of organs available for transplant. There is a need for alternative therapies for liver diseases. The transplantation of isolated hepatocytes (liver cells) has been used as an experimental therapy for liver disease in a limited number of cases. Recently, the 100th case of hepatocyte transplantation was reported. This review discusses the history of the hepatocyte transplant field, the major discoveries that supported and enabled the first hepatocyte transplants, and reviews the cases and outcomes of the first 100 clinical transplants. Some of the problems that limit the application or efficacy of hepatocyte transplantation are discussed, as are possible solutions to these problems. In conclusion, hepatocyte transplants have proven effective particularly in cases of metabolic liver disease where reversal or amelioration of the characteristic symptoms of the disease is easily quantified. However, no patients have been completely corrected of a metabolic liver disease for a significant amount of time by hepatocyte transplantation alone. It is likely that future developments in new sources of cells for transplantation will be required before this cellular therapy can be fully implemented and available for large numbers of patients.

In vitro human hepatocyte-based experimental systems for the evaluation of human drug metabolism, drug-drug interactions, and drug toxicity in drug development.

Species difference in drug metabolism and drug toxicity is a well-established phenomenon. As a result, the classical paradigm of preclinical testing of drug candidates in animals may not be appropriate. One preclinical approach to evaluate human drug properties, especially ADMET (absorption, disposition, metabolism, elimination, and toxicity) properties, is to apply in vitro experimental systems with relevant human properties. The latest advances include the use of human hepatocytes to evaluate hepatic uptake, metabolism, efflux and toxicity. Successful cryopreservation of human hepatocytes to retain high viability, metabolic capacity, as well as the ability to be cultured allow routine application of this relevant experimental system. This review summarizes the latest findings on human hepatocytes isolation, cryopreservation, culturing, as well as application in the evaluation of metabolic stability, metabolite profiling, hepatic uptake and efflux, metabolic drug-drug interactions, and drug toxicity. The use of hepatocyte to evaluate the role of metabolism in drug toxicity represents a major advance in drug toxicity evaluation. The use of the novel integrated discrete multiple organ coculture (IdMOC) system allows the evaluation of the role of hepatic metabolism on nonhepatic toxicity.

Biomarkers and Human Hepatocytes

The accuracy of preclinical safety evaluation to predict human toxicity is hindered by species difference in drug metabolism and toxic mechanism between human and nonhuman animals. In vitro human-based experimental systems allowing the assessment of human-specific drug properties represent a logical and practical approach to provide human-specific information. An advantage of in vitro approaches is that they require only limited amounts of time and resources, and, most importantly, do not invoke harm to human patients. Human hepatocytes, with complete hepatic metabolizing enzymes, transporters and cofactors, represent a practical and useful experimental system to assess drug metabolism. The use of human hepatocytes to evaluate two major adverse drug properties, drug-drug interactions and hepatotoxicity, are reviewed. The application of human hepatocytes in metabolism-based drug-drug interactions includes metabolite profiling, pathway identification, CYP450 inhibition, CYP450 induction, and uptake and efflux transporter inhibition. The application of human hepatocytes in toxicity evaluation includes in vitro hepatotoxicity and metabolism-based drug toxicity determination. Correlation of drug toxicity with proteomics and genomics data may allow the discovery of clinical biomarkers for early detection of liver toxicity.

An algorithm that predicts the viability and the yield of isolated human hepatocytes from surgical remnant tissues

Introduction: The liver is an organ that carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of hepatocytes is especially important in the fields of pharmacokinetics, toxicology and translational research. In order to obtain hepatocytes suitable for use as a model, hepatocytes with high viability and yield must be isolated. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Methods: Remnant liver pieces from tissue designated as surgical waste was collected from 1034 donors with informed consent. Corresponding data on donor characteristics, medical histories, operations, tissue processing and cell isolation parameters were collected and entered into a database. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications. Hepatocyte yield and viability were then subsequently determined using a hemocytometer-based trypan blue exclusion assay. Univariate and multivariate analyses were carried out using Software R. Results: Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. Discussion: In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified as candidates for isolating hepatocytes. These isolated hepatocytes can be then used for studying cellular metabolism, pharmacokinetics, toxicology of xenobiotics, translational research etc. The use of human hepatocytes also remains an important model for validating research done in animal models and can also reduce the number of animals used for research in line with the 3R ethical framework. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen (1). Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

HUMAN HEPATOCYTE INDUCTION STUDIES TO EVALUATE POTENTIAL OF CYP3A4 INDUCTION IN VIVO: TWENTY COMPOUNDS WITH THREE DONORS

Recent guidance from regulatory agencies advocates the use of human hepatocytes as the test system for evaluating cytochrome P450 induction potential of drug candidates. The use of hepatocytes which have been prequalified for response to known clinical inducers and non‐inducers are indicated. In addition, the use of mRNA alone is suggested as the necessary endpoint to evaluate induction response. However, the applicability of using prequalified hepatocytes as the test system, and the use of mRNA alone as a response endpoint, has not been widely demonstrated. To this end, we have examined the induction response from a set of 20 model compounds which includes established clinical inducers and non‐inducers of CYP3A4/5, in cryopreserved hepatocytes from three donors. Endpoints examined were CYP3A4 mRNA and testosterone 6ß‐hydroxylase activity, typically over 8 concentration points to enable calculation of EC50 and Emax. Results were used to determine relative induction scores (RIS), R3 values, and fold‐induction threshold values that may classify a drug candidate as a potential clinical inducer. The results and merits of these and other parameters for evaluating induction potential of drug candidates will be discussed.

Institutional Profile: Karolinska Institutet

Research in pharmacogenomics has been intensive at Karolinska Institutet (KI) for approximately 25 years. Initial initiatives were focused on the identification and characterization of novel CYP2D6 alleles causing ultrarapid or defective drug metabolism. Such discoveries were possible owing to the early implementation of therapeutic drug monitoring and the access to individuals phenotyped with respect to drug metabolism. The translational work at KI has been of utmost importance for successful research, including functional characterization and clinical validation of allelic variants in drug metabolism, as well as discoveries of novel polymorphisms, recent examples being the CYP2C19 and UGT2B17 genes. The clinical pharmacology laboratory at KI campus Huddinge is one of the leading sites for therapeutic drug monitoring in northern Europe and obtains an increasing number of clinical requests, also important for pharmacogenetic research. Furthermore, the recently opened Center for Hematology and Regenerative Medicine, with a clear translational emphasis, offers an opportunity for studying drug metabolism and toxicity in vitro by use of human hepatocytes.

Determination of Time-Dependent Inactivation of CYP3A4 in Cryopreserved Human Hepatocytes and Assessment of Human Drug-Drug Interactions

Assessment of time-dependent inhibition (TDI), especially CYP3A4, is an important parameter for preclinical and clinical development. The use of human liver microsomes (HLM) is the most common in vitro matrix to assess TDI, but this often leads to an overprediction of an actual effect observed clinically. Recently, the use of human hepatocytes has been hypothesized as a more relevant and possibly predictive matrix for the assessment of CYP3A4 TDI. Our work evaluates and optimizes three different human hepatocyte assays for the assessment of CYP3A4 TDI using pooled cryopreserved human hepatocytes. Using two of the optimized methods, the time-dependent inhibition kinetic parameters (K(I) and k(inact)) for four known CYP3A4 TDI (diltiazem, erythromycin, verapamil, and troleandomycin) were determined. When comparing TDI in HLM, the K(I) values from hepatocytes were in general 4- to 13-fold higher than that in HLM, whereas the k(inact) values in human hepatocytes were similar or slightly higher or lower depending on the inhibitor. The inactivation potency (k(inact)/K(I)) for four tested CYP3A4 inactivators in human hepatocytes was generally lower than that in HLM due to either lower affinity (K(I)) or lower inactivation rate (k(inact)) or both. When drug interactions were simulated with Simcyp using either HLM or human hepatocyte data, the predictions using the kinetic parameters from human hepatocytes resulted in a much better simulated change in pharmacokinetics compared with observed clinical data.

The Impact of Hepatic Uptake on the Pharmacokinetics of Organic Anions

The disposition of seven marketed and two AstraZeneca acid (organic anion) compounds with a range of volume of distribution at steady state (V(ss)) and clearance have been profiled in rat and dog. Pharmacokinetic (PK) parameters along with liver and muscle tissue levels were collected, and their contributions to total V(ss) were calculated. The physiologically based prediction of V(ss) correlated (all predictions within 2-fold) with the V(ss) obtained from plasma PK analysis. The V(ss) of the acid drugs with atypically high values could be explained by significant sequestering of compound to the liver. A "media loss" in the in vitro hepatocyte assay that monitors loss of compound from the incubation media along with physiologically based PK (PBPK) modeling was assessed for its ability to accurately predict the impact of hepatic uptake on both clearance and V(ss). This methodology significantly improved the prediction of metabolic in vivo clearance compared with standard hepatocyte scaling approaches that do not take into account hepatic uptake. Predictions of V(ss) from the media loss assay also correlate with the measured values from plasma PK analysis. However, hepatic uptake will have little overall impact on half-life, because of the concomitant impact on both Cl and V(ss), as long as hepatic extraction is not high. The methodology described here is particularly useful when there is no allometric relationship between species as a result of interspecies differences in liver uptake. In this situation, the potential use of human hepatocytes combined with PBPK modeling avoids the question of which species pharmacokinetics is most predictive to humans.

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

Human hepatocytes in primary culture are a very useful model to directly assess induction of gene expression by xenobiotics. We developed a cytochrome P450 (P450) activity cocktail assay using model substrates for the seven important P450s 1A2 (phenacetin), 2B6 (bupropion), 2C8 (amodiaquine), 2C9 (tolbutamide), 2C19 (S-mephenytoin), 2D6 (propafenone), and 3A4 (atorvastatin). Metabolite formation was determined by liquid chromatography-tandem mass spectrometry in hepatocyte culture supernatants. Atorvastatin has not been previously assessed as a CYP3A probe drug. We demonstrate highly selective atorvastatin ortho-hydroxylation by CYP3A4 by recombinant P450s. In human liver microsomes ortho-hydroxyatorvastatin formation was highly correlated with CYP3A4 protein content (r(s) = 0.78, p < 0.0001, n = 150). We profiled induction of these P450 activities in primary human hepatocytes after treatment with 30 microM atorvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin for 24 to 72 h. Except for pravastatin, all statins induced P450 activities to various degrees, approximately in the order atorvastatin > simvastatin > lovastatin > rosuvastatin. Inducibility of P450s followed the order CYP2C8 > CYP3A4 > CYP2C9 > CYP2B6 > CYP2C19 approximately CYP2D6 > CYP1A2. The strongest induction was observed for amodiaquine N-desalkylation, which was induced approximately 20-fold. Quantitative reverse transcription-polymerase chain reaction confirmed corresponding changes on the mRNA level with even more dramatic induction up to almost 100-fold. These data suggest a broader inducing effect of statins on cytochrome P450s and possibly other absorption, distribution, metabolism, and excretion genes than previously known, thus further emphasizing their drug-drug interaction potential. Our cocktail assay should be helpful for economical use of human hepatocytes in the assessment of P450 induction by drugs and drug candidates.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Human and animal hepatocytes in vitro with extrapolation in vivo

Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and “Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.

A Survey of Consumer Attitudes to the Supply and Use of Human Hepatocytes in the United Kingdom

Human hepatocytes are the model of choice for pharmacotoxicological studies, but their acquisition is often problematic due to ethical and logistical difficulties. The UK Human Tissue Bank is a not-for-profit organisation that acquires and processes human tissue, with a specialist interest in the isolation of human hepatocytes. A recent in-house survey of the processing of liver tissue over 1 year revealed that freshly isolated hepatocytes were underutilised due to mismatched consumer demand, despite the published need for them. We present the results of a telephone survey to investigate the reasons behind this paradox. This survey highlighted some problem areas, including "out of hours" availability of cells and personnel difficulties, but overall, demonstrated the value of such a service, with numerous researchers taking advantage of available good quality human hepatocytes. Although further work is required in optimising long-term storage protocols through cryopreservation, we have demonstrated that tissue handling of this type can be successful and beneficial to the pharmaceutical and biotechnology industries.

Human Hepatic Cell Cultures: In Vitro and In Vivo Drug Metabolism

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.

Generation of hepatocyte-like cells from human embryonic stem cells.

Use of human hepatocytes for therapeutic and drug discovery applications is hampered by limited tissue source and the inability of hepatocytes to proliferate and maintain function long term in vitro. Human embryonic stem (hES) cells are immortal and pluripotent and may provide a cell source for functional human hepatocytes. We report here that hES cells can be induced to differentiate into hepatocyte-like cells. Treatment with sodium butyrate induced hepatic differentiation as well as significant cell death, resulting in approximately 10-15% yield of a homogeneous population of cells. The differentiated cells have morphological features similar to that of primary hepatocytes and 70-80% of the cells express liver-associated proteins (albumin, alpha-1-antitrypsin, cytokeratin 8 and 18), accumulate glycogen, have inducible cytochrome P450 activity, and do not express alpha-fetoprotein. Because of the inherent proliferative capacity of hES cells, these cells may provide a reliable source of normal human hepatocytes for research and transplantation.

In vitro characterization of porcine hepatocyte function.

The clinical consequences of acute liver failure are associated with high mortality. Intensive medical intervention is required to treat the symptoms of liver failure, including coagulopathy, metabolic instability, and encephalopathy. Providing temporary liver support with an extracorporeal liver assist device could stabilize the patient until a donor liver became available or the patient's own liver was able to recover. The use of human hepatocytes as the biologic component of the assist device is precluded by the scarcity of available tissue and the limited proliferative potential of adult hepatocytes in vitro. Consequently, porcine hepatocytes are being evaluated as a cell source for liver assist devices. Maintaining differentiated function in isolated hepatocytes, however, remains a challenge in the development of this technology and is complicated by the fact that the key therapeutic functions for short-term survival have not been well defined. Several approaches have been effective in prolonging rodent hepatocyte function in vitro, including manipulation of extracellular matrix. Here, we have investigated porcine hepatocyte function in vitro with a specific emphasis on the response to exogenous collagen matrix. In control cultures, albumin secretion increased during the first 7-10 days of culture to an average of 50 +/- 17 microg/day/10(6) cells and then decreased over the next 2 weeks. The pattern of urea synthesis was slightly different in that it was highest in the first 1-3 days postisolation (140 +/- 19 microg/day/10(6) cells) and then decreased by about 50% to a plateau level that was stable during the next 3-4 weeks of culture. Cytochrome P450-mediated activities were the most labile with time in culture and were undetectable after the first week in the absence of pharmacological inducers. In contrast to results reported for rat cells, porcine hepatocytes exhibited differentiated function in the absence of any modification of the culture dish surface and function was not increased or prolonged in the presence of exogenous collagen.

Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells

Loss of cytochrome P-450 content is a common feature in conventional culture systems of primary hepatocytes. In contrast to the standard in vitro situation, in vivo each hepatocyte is exposed to an extracellular matrix (space of Disse) at two opposing basolateral surfaces. This in vivo symmetry has been reconstructed in vitro by culturing rat or human hepatocytes within two layers of collagen, thus forming a sandwich configuration. Activation of dimethylbenzanthracene (DMBA) or benzo[ a]pyrene (BaP) was studied in rat and human hepatocytes. Genotoxic effects were studied in a three-dimensional co-culture model between sandwich hepatocytes and mammalian cells using the comet assay for detection of DNA strand breaks, and the HPRT test for detection of gene mutations. Sandwich hepatocytes generated active metabolites. The maintenance of metabolic properties in hepatocytes was dependent on extracellular matrix geometry. The number of DMBA- or BaP-induced genotoxic effects tended to be higher than in standard S-9 mix assays. While the ability to activate indirect carcinogens disappears within hours in primary hepatocytes, hepatocyte sandwich cultures enhance their ability to activate indirect carcinogens within 1 wk and retain this activity for up to 2 wk. This is the main advantage of the sandwich method over the more simple and conventional assays. While freshly isolated hepatocytes, regardless of whether in sandwich culture or in conventional assays, are injured by the isolation procedure and possess a corresponding reduced activation ability, hepatocytes in sandwich cultures recover over the course of a few days, and acquire a much higher ability to activate indirect carcinogens. Consequently, the indirect carcinogens BaP and DMBA, which were ineffective (BaP) or exhibited only weak effects (DMBA) at a concentration of 160 nmol/ml in 1–2-day-old hepatocytes, were clearly effective (BaP) or showed about a threefold increase in genotoxicity (DMBA) in 8-day-old hepatocytes in sandwich co-culture. In contrast to the experiments with S-9 mix, which is toxic to mammalian cells and does not allow treatment times of more than 2–3 hr, cells in co-culture with human or rat hepatocytes can be treated for at least 24 hr. The use of sandwich cultures has not yet been described for genotoxicity studies. The results of the present study may perhaps facilitate the acceptance of this method as a co-culture model for the field of genetic toxicology. Use of hepatocytes alone for genotoxicity studies cannot be recommended for difficulties in isolating intact cells from the sandwich cultures. The use of human hepatocytes in sandwich co-culture should enable a more relevant evaluation of potential human genotoxicity with specific chemicals and should put the extrapolation of genetic toxicology data from animal species to humans on a more scientific basis. Beyond that, experiments with animals in vivo could be avoided.