The emergence of tissue engineering raises new possibilities for the study of complex physiological and pathophysiological processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. We discuss here some of the 'design principles' for recreating the interwoven set of biochemical and mechanical cues in the cellular microenvironment, and the methods for implementing them. We emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.

Capturing complex 3D tissue physiology in vitro.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Tissue function depends on hierarchical structures extending from single cells ( approximately 10 microm) to functional subunits (100 microm-1 mm) that coordinate organ functions. Conventional cell culture disperses tissues into single cells while neglecting higher-order processes. The application of semiconductor-driven microtechnology in the biomedical arena now allows fabrication of microscale tissue subunits that may be functionally improved and have the advantages of miniaturization. Here we present a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks. The need for such models is underscored by the high rate of pre-launch and post-market attrition of pharmaceuticals due to liver toxicity. We demonstrate utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins. The combination of microtechnology and tissue engineering may enable development of integrated tissue models in the so-called 'human on a chip'.

Microscale culture of human liver cells for drug development

Of the thousands of novel compounds that a drug discovery project team invents and that bind to the therapeutic target, typically only a fraction of these have sufficient ADME/Tox properties to become a drug product. Understanding ADME/Tox is critical for all drug researchers, owing to its increasing importance in advancing high quality candidates to clinical studies and the processes of drug discovery. If the properties are weak, the candidate will have a high risk of failure or be less desirable as a drug product. This book is a tool and resource for scientists engaged in, or preparing for, the selection and optimization process. The authors describe how properties affect in vivo pharmacological activity and impact in vitro assays. Individual drug-like properties are discussed from a practical point of view, such as solubility, permeability and metabolic stability, with regard to fundamental understanding, applications of property data in drug discovery and examples of structural modifications that have achieved improved property performance. The authors also review various methods for the screening (high throughput), diagnosis (medium throughput) and in-depth (low throughput) analysis of drug properties. * Serves as an essential working handbook aimed at scientists and students in medicinal chemistry * Provides practical, step-by-step guidance on property fundamentals, effects, structure-property relationships, and structure modification strategies * Discusses improvements in pharmacokinetics from a practical chemist's standpoint

Drug-Like Properties: Concepts, Structure Design and Methods from ADME to Toxicity Optimization

Drug-induced liver injury is a frequent cause of hepatic dysfunction Reliably establishing whether the liver disease was caused by a drug requires the exclusion of other plausible causes and the search for a clinical drug signature The drug signature consists of the pattern of liver test abnormality, the duration of latency to symptomatic presentation, the presence or absence of immune-mediated hypersensitivity and the response to drug withdrawal Determination of causality also includes an evaluation of individual susceptibility to drug-induced liver injury This susceptibility is governed by both genetic and environmental factors Components of the drug signature in conjunction with certain risk factors have been incorporated into formal scoring systems that are predictive of the likelihood of drug-induced liver injury The most validated scoring system is the Roussel-Uclaf causality assessment method, which nonetheless retains certain imperfections Mitigating the potential for drug-induced liver injury is achieved by the identification of toxicity signals during clinical trials and the monitoring of liver tests in clinical practice There are three signals of liver toxicity in clinical trials: (i) a statistically significant doubling (or more) in the incidence of serum alanine aminotransferase (ALT) elevation >3 x the upper limit of normal (ULN); (ii) any incidence of serum ALT elevation >8-10 x ULN; and (iii) any incidence of serum ALT elevation >3 x ULN accompanied by a serum bilirubin elevation >2 x ULN Monitoring of liver tests in clinical practice has shown unconvincing efficacy, but where a benefit-risk analysis would favour continued therapy, monthly monitoring may have some benefit compared with no monitoring at all With rare exception, treatment of drug-induced liver injury is principally supportive Drug toxicity is the most common cause of acute liver failure, defined as a prolonged prothrombin time (international normalised ratio > or =15) and any degree of mental alteration occurring <26 weeks after the onset of illness in a patient without pre-existing cirrhosis A patient who meets these criteria must be evaluated for liver transplantation The pathogenesis of drug-induced liver injury can be examined on the basis of the two principal patterns of injury The hepatocellular pattern is characterised by a predominant rise in the level of transaminases and results from the demise of hepatocytes by means of either apoptosis or necrosis The cholestatic pattern is characterised by a predominant rise of the serum alkaline phosphatase level and usually results from injury to the bile ductular cells either directly by the drug or its metabolite, or indirectly by an adaptive immune response

Drug-Induced Liver Injury

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures.

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New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures.