Hepatocyte s

Methods and Protocols

Edited by
Chapter 1 General Review on In Vitro Hepatocyte Models and Their Applications Christiane Guguen-Guillouzo and Andre Guillouzo Abstract In vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new welldifferentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity

PatrickMaurel
INSERM, Universit Montpellier 1, and Institut de Recherche en Biothrapie,

of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with highcontent imaging and in toxicogenomics technologies. Key words: Hepatocytes, liver cell lines, HepaRG cells, stem cells, culture conditions, cryopreservation, differentiation, proliferation, bile metabolism, xenobiotic metabolism, transporters, hepatotoxicity, toxicotranscriptomics, high-content imaging, hepatocyte therapies, virology, parasitology. 1. Introduction The technique of high-yield preparation of isolated hepatocytes by collagenase perfusion was published in 1969 by Berry and Friend (1) and the two-step procedure that is now the usual P. Maurel (ed.), Hepatocytes, Methods in Molecular Biology 640, DOI 10.1007/978-1-60761688-7_1, Springer Science+Business Media, LLC 2010 Guguen-Guillouzo and Guillouzo way to get hepatocyte suspensions was introduced by Seglen in 1972 (2). One year later Bissell et al. (3) described the rat hepatocyte monolayer cultures and in 1982 high-yield preparation and primary culture of adult human hepatocytes were reported by Guguen-Guillouzo et al. (4). During 40 years an extraordinary number and diversity of studies have been carried out with isolated hepatocytes from livers of humans and various animal species, dealing with both hepatocyte functions and

applications in diverse fields. Many reviews and multiauthored books have already covered many of these topics, e.g. (5, 6). However, continuous progress is being made with isolated hepatocytes that deserves periodic review. In this chapter, we first summarize the main experimental conditions presently used to maintain functional hepatocytes in vitro and then attempt to analyze the more recent findings in the biology of the hepatocyte and the major application fields. Through this chapter the performance of isolated hepatocytes in suspension or in primary culture will be challenged with that of the other in vitro liver cell models, especially new established liver cell lines. 2. Culture Conditions of Hepatocytes 2.1. Primary Adult Hepatocytes Hepatocytes can be obtained from whole liver or wedge fragments. Today human hepatocytes are marginally isolated from livers unsuitable for liver transplantation and mostly from liver fragments resected from primary or secondary tumors or some other liver diseases. Freshly isolated hepatocytes exhibit the typical structure and most of the functions of their in vivo counterparts but they have lost specialized membrane domains such as intercellular junctions and bile canaliculi and they do not survive for more than a few hours in suspension. To survive longer they must attach to a substratum. When plated in conventional culture conditions, they

reaggregate and reconstitute bile canaliculuslike structures but they exhibit early phenotypic alterations andsurvive for only a few days. In agreement with these changes, deregulation of a large set of genes was observed by comparing suspended and attached primary human hepatocytes using the microarray approach (7). In addition to their scarce and unpredictable availability and interdonor variability, human hepatocytes behave differently upon their rodent counterparts. Indeed, at least for some functions such as cytochromes P450 (CYP), especially CYP1A2 and CYP3A4, an early and marked drop followed by a Hepatocyte Models and Applications transient increase is frequently observed supporting the view that they are more active 23 days after plating. Extensive reviews have been published on conditions to improve hepatocyte survival and function in vitro (813). Very early it appeared that several factors were critical for survival and function of hepatocytes in primary culture, they included soluble factors (i.e., medium composition) and pericellular environmental factors (matrix proteins as well as other cell types). Addition of 2% dimethylsulfoxide (DMSO) (14) and other chromatin remodeling agents such as trichostatin A (15), the use of the sophisticated Lanfords medium at least for monkey and human hepatocytes (16), the use of extracellular matrices that prevent cell spreading such as matrigel (17) and the sandwich configuration (18), and cocultivation with nonparenchymal cells (19, 20) are today among the most convincing culture conditions for extended survival of functional hepatocytes. Various other models have been designed, including

bioreactors providing scaffolds for the cells that can be continuously oxygenated and perfused. It is also well established that entrapping in collagen or alginate gels allows hepatocytes to survive for several days instead of a few hours in suspension (21). Recently, high HCV replication was obtained in primary human hepatocytes maintained well differentiated by the use of appropriate culture conditions consisting in seeding cell suspensions (>95% viability and low-apoptotic activity) at high density (1.8 million/60 cm2 plate) in plates treated with polylysine coated with a 3-D specific rat tail collagen 1 matrix, in a medium containing 20% fetal calf serum during spreading only Suspended hepatocytes can be stored for either a short period (13 days) in hypothermic conditions (05C) or prolonged periods in liquid nitrogen (23, 24). Even when using well-defined freeze/thaw conditions nearly half of cryopreserved suspended hepatocytes lose their ability to attach to plastic after thawing. However, when the cells are first encapsulated or entrapped, e.g., in alginate gels, viability is well maintained after cryopreservation and CYP activities are comparable to those measured in fresh hepatocyte monolayer cultures (25, 26). A further improvement could come by vitrifying encapsulated hepatocytes. Hepatocyte Cell Lines Hepatocyte cell lines can be obtained by oncogenic immortalization or from tumors. A lot of efforts have been put on oncogenicimmortalization of adult hepatocytes but the results are

quite disappointing. Immortalized cells tend to be genetically unstable and lose their phenotypic characteristics. However, combination of SV40 T and TERTmediated gene transfections to produce genetically stable cells (28) deserves further investigation. Only few immortalized liver cell lines expressing some liverspecific functions have been described. Probably the most powerful immortalized cell line is the Fa2N-4 cell line originated from human hepatocytes transfected with the SV40 large T antigen gene (29). These cells express various drug-metabolizing enzymes, including some major CYPs and transporters. However, the nuclear constitutive androstane receptor (CAR) and several transporters are >50-fold lower than in primary human hepatocyte cultures and low if any response of CYP2B6 and CYP3A4 was evidenced with CAR activators (30). The most used human hepatocyte cell lines (e.g., HepG2, Hep3B, PLC/PRFs Huh7, HBG) are derived from tumors. Since its initial establishment (31) the HepG2 cell line has lost a substantial and variable set of liverspecific functions, especially the major CYPs involved in xenobiotic metabolism. 3. Applications to the Biology of the Hepatocyte 3.1. Hepatocyte Differentiation 3.1.1. Commitment and Stability of the Hepatocyte Phenotype During the last 15 years, isolated hepatocytes have been extensively used for

studies on the regulation of liver-specific genes. Primary cultures have been very useful for the understanding of the role of these genes on tissue function specificity as well as the role of environmental factors on their regulation. Several transcription factor families that govern tissue-restricted gene expression were clearly identified; structurally related DNA-binding domains and include the variant homeodomaincontaining proteins (HNF-1, HNF-1); the winged helix family proteins HNF-3, , and (also called FoxA1, 2, and 3); members of the nuclear hormone receptor family (HNF-4, COUP-TFII, LRH-1, FXR, and PXR); the basic leucine zippercontaining factor C/EBP; and the onecut homeodomain protein HNF-6 (48). The genes found to be targeted by HNF1 in primary human hepatocytes encode products whose functions represent a substantial cross section of hepatocyte biochemistry. HNF1 contributes to the transcriptional regulation of many of the central rate-limiting steps in gluconeogenesis and associated pathways. HNF1 also binds to genes whose products are central to normal hepatic function, including carbohydrate synthesis and storage, lipid metabolism (synthesis of cholesterol and apolipoproteins), detoxification (synthesis of cytochrome P450 monooxygenases), and synthesis of serum proteins (albumin, complements, and coagulation factors). HNF1 also regulates primarily hepatocyte polarization (49). Meanwhile, notch-2 exerts a critical role in the cell fate of hepatic bipotent progenitors (36) and HNF4 is a key regulator of morphological and functional differentiation of hepatocytes essential for the formation of a polarized hepatic epithelium (50) and cellcell contactsCross-regulatory cascades between

hepatic transcription factors have been implicated in the commitment of the hepatic phenotype. Analysis of recruitment to regulatory regions of the main hepatic regulators during liver development has revealed a gradual increase in complexity of autoregulatory and cross-regulatory circuits (52). As a consequence, none of these factors is expressed exclusively in adult hepatocytes and none of them can induce alone the hepatic program in nonhepatic cells, and transcriptional regulation of most of the liver-specific genes requires a combinatorial action of the above activators. This has been confirmed from a recent genome-wide promoter occupancy study in liver which concludes that >40% of the promoters of active genes were bound by HNF-4, and most of the promoters bound by HNF-1 or HNF-6 were also occupied by HNF-4 (53). In addition, as in vivo, extracellular signals contribute to regulating these transcription factors during hepatic differentiation in vitro. They also coordinately contribute to stabilize their activities in differentiated hepatocytes in primary culture, as shown by Liu et al. (54) for c/EBPs. This high complexity that characterizes liver parenchymal cells explains for a part why it is difficult to preserve a high stability of liver-specific functions in hepatocytes in vitro and why attempts in restoring a transcription factor activity by gene transfection in hepatic cell lines as assayed by several investigators failed to stably restore an extinguished liver function in these permanent cell lines (55). However, transient overexpression or extinction by transfection strategy into primary hepatocytes or Hepatocyte Models and Applications 9 hepatoma cell lines provide

very useful approaches for highlighting the role of these genes in specific living cells, which constitute a very efficient alternative to transgenic animals. Conditions mimicking environmental signals including extracellular matrix component deposition or establishment of intercellular communications between hepatocytes and other hepatic cells such as primitive biliary cells and liver endothelial cells were successfully developed in order to support stability of liver-specific functions for a prolonged period (56, 57). However, although induction of several functions is possible for 34 weeks under exposure to appropriate inducers and using these coculture models, the level of gene transcription activity remains less than half of that in freshly isolated hepatocytes (57). In addition, cells undergo gradual progression to aging. This contrasts with the highly differentiated hepatic HepaRG cells which can reverse to undifferentiated progenitors with restored proliferative activity at low cell density. For this reason these cells constitute a unique model for understanding the mechanisms which allowhem to escape from aging and death and restore their progenitor cell properties. The major property is the commitment signal making progenitors able to undergo a complete bipotent hepatoblast differentiation program up to mature hepatocytes for one lineage and to biliarylike cells for the other. Indeed, distinct steps with sequential transcription factor expression have been reported along the hepatocyte differentiation process (36). Gene factors controlling this reprogramming are not known yet. Importent modulators could be involved, such as p53, p21 and Rb. Noteworthy,

these genes and wt- catenin are normally regulated in HepaRG cells.

References

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