Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis? (original) (raw)
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Archives of Toxicology, 2013
Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed. Keywords Drug safety testing Á In vitro models Á Primary hepatocyte cultures Á Dedifferentiation Á Epigenetics Á Histone deacetylases Á DNA methyltransferases Á Liver-enriched transcription factors Á MicroRNA Currently available hepatocyte-based in vitro models in safety testing of new drug candidates
Towards an Extended Functional Hepatocyte In Vitro Culture
Tissue Engineering Part C-methods, 2009
Hepatocyte cultures, in general, show loss of biochemical function, such as reduction of protein synthesis or loss of cytochrome P450 enzyme activity. Among the many reasons that may account for this reduction of hepatocyte function in vitro are the hepatocyte adaptation to the culture conditions and the withdrawal from their natural environment. In the present study, alternative cell culture models were used, attempting to increase liver-specific functions in vitro. Namely, methods that allow the provision of 3D architectures of rat hepatocytes in vitro that better mimic some liver characteristics, are preserved. A model of hepatocyte aggregates (3D) culture in stirred tanks, that allow maximized metabolic hepatocyte capacity, was adopted and compared with the conventional well plates. Herein, the rat liver has been disaggregated undergoing a perfusion step, and an additional Percoll step was introduced to enrich the hepatocyte population in the cultures. The effect of different inocula concentrations, impeller types and culture media on the culture was tested. Both general culture parameters, such as LDH, glucose, lactate levels, as well as liver cellspecific functions, such as albumin and urea production and CYP450 activity (ECOD and testosterone assay) have been assessed. Our preliminary results suggest that 3D cultures exhibit stronger liver-specific functions than cells in monolayers, for a longer period of time, thus indicating that the former type of culture may be a promising approach for drug screening.
Drug Metabolism Reviews, 2003
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 4 hr. 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% CO 2 . 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 system is 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 for 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.
Recent advances in human hepatocyte culture systems
The liver fulfills many vital processes in mammals. It is the central organ of energy metabolism, biotransformation of xenobiotics, and synthesis of plasma proteins under physiological and pathophysiological conditions. Primary cultures of hepatocytes have been and still are an important tool to study liver-specific processes and functions. So far, most of the studies with hepatocyte cultures have primarily been performed with rat hepatocytes. However, interspecies differences in all aspects of hepatocyte function exist and are recognized and investigated since more than 30 years: Differences have been reported for the intracellular distribution of gluconeogenic enzymes (1-4), for apolipoprotein A-IV expression (5), metabolic regulation of cholesterol and triacylglycerol synthesis (6), and cytochrome P450 induction . Therefore, primary human hepatocytes should be the system of choice for the evaluation of liver specific processes in human: (i) the biology of human viral pathogens or parasites and (ii) drug metabolism in phase I and II reactions (9, 10).
Archives of toxicology, 2016
The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantif...
Long-term culture and coculture of primary rat and human hepatocytes
Methods in molecular biology (Clifton, N.J.), 2013
The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity or viral infection is a major cause of death in the United States. The development of Bioartificial Liver (BAL) devices and the demand for pharmaceutical and cosmetic toxicity screening require the development of long-term hepatocyte culture techniques. However, primary hepatocytes rapidly lose their cuboidal morphology and liver-specific functions over a few days in culture. Accumulation of stress fibers, loss of metabolic function, and cell death are known phenomena. In recent years, several techniques were developed that can support high levels of liver-specific gene expression, metabolic and synthetic function for several weeks in culture. These include the collagen double-gel configuration, hepatocyte spheroids, coculture with endothelial cells, and micropatterned cocultures with 3T3-J2 fibroblasts. This chapter covers the current status of...
Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements
Bioengineering
Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the “sandwich cultures”, were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, lif...
Long-term culture of functional hepatocytes
Toxicology in Vitro, 1990
Abstraet--R.ecent studies have clearly demonstrated that the hepatocyte requires a complex and well defined environment to survive and maintain differentiated functions in vitro. Soluble factors as well as cell-matrix and cell-well interactions have been found to affect markedly hepatocyte functions. Thus co-culturing hepatocytes with another rat liver cell type results in a prolonged expression of liver functions including phase I and phase II drug-metabolizing enzymes. Addition of corticosteroids to the co-culture medium is a prerequisite, and accumulation of insoluble matrix components is observed within a few days primarily between the two cell types. Hepatocyte cultures have been widely used for pharmacology and toxicology studies during recent years, but most studies deal with short-term investigations. Although specific functions are not completely stabilized the use of long-term hepatocyte cultures represents a promising tool to investigate enzyme induction and inhibition, and drug chronic toxicity.