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Hepatocyte Users Group Meeting


 
 

OC01 - FROM GENE OVEREXPRESSION TO CRE/LOX GENE TARGETING: ADENOVIRUS VECTORS AS AN EFFICIENT TOOL TO STUDY GENE FUNCTION IN PRIMARY HEPATOCYTES. 

S Prost, University of Edinburgh, Pathology. 

The analysis of a gene function usually involves the study of its overexpression and, where possible, the study of the consequences of its deletion. Both types of study can benefit from the use of adenovirus vectors in vitro.

For expression studies, transient transfection of expression plasmids is usually performed using lipid-based compounds. The efficiency of transfection is often poor, the expression of the gene late and transient. By contrast adenovirus infection of primary hepatocytes is very reliable, highly efficient (more than 95 % infected cells), expression is rapid and stays stable for longer (at least 96 hours). No effect is observed on hepatocytes health and viability, even when cells are subsequently treated with damaging agents such as UV.

Regarding gene deletion studies, the development of the Cre/Lox system, is a milestone in conditional gene targeting. In this approach, Cre-recombinase excises a gene flanked by LoxP recognition sequences. However, many problems remain to achieve successful tissue specific, temporally controlled gene excision in vivo.

By contrast, adenovirus-mediated deletion of floxed alleles in primary hepatocytes is a straightforward and highly efficient method for conducting studies of conditional gene targeting. It allows baseline data and principles to be established in a regulated setting without the drawbacks on in vivo work. 

OC02 - ACTIVATION OF SIGNALLING PATHWAYS DURING HEPATOCYTE ISOLATION & CULTURE AND THE LOSS OF LIVER FUNCTION EX VIVO 

Alan J Paine, Molecular & Cellular Toxicology Research Group, Department of Pharmacy, King’s College London, London, SE1 9NN, U.K. 

A major limitation to the use of primary cultures of hepatocytes in biomedical research is their rapid loss of liver specific function. For example, the transcription of albumin is silenced during the well-established hepatocyte isolation procedure with collagenase while liver specific cytochrome P450 (CYP) mRNAs are lost, later, during the first few hours of culture.

The different responses of albumin and CYP gene expression during hepatocyte isolation and culture has led us to propose that two distinct processes underlie the loss of liver function ex vivo especially as the loss of CYP mRNAs is highly correlated with hepatocyte shape changes and induction of ribonuclease activity.

A much earlier change in hepatocyte gene expression is the activation of NF-B, a mediator of the hepatic acute phase response, just 10 minutes into hepatocyte isolation. Since NF-B can be activated by several signalling cascades/stimuli, e.g. MAP kinases (MAPK), nitric oxide synthases and hypoxia, we have examined the effects of hepatocyte isolation on these processes. Although p42/44 MAPKs are activated in the donor organ prior to the addition of collagenase and the response of other MAPKs is very different in liver perfusions with and without collagenase the clear “take-home message” is that hypoxia underlies the activation of NF-B during liver cell isolation.

In conclusion, greater appreciation of events underlying the two distinct, early, time-frames during hepatocyte isolation and culture represents the surest foundation for the successful application of hepatocyte cultures in biomedical research rather than relying on empirical manipulations of hepatocyte culture conditions once their differentiated phenotype has been lost. 
 

 

OC03 -EFFECT OF TRICHOSTATIN A, A HDAC INHIBITOR, ON MAINTENANCE OF HEPATIC FUNCTIONS IN PRIMARY CULTURES OF RAT HEPATOCYTES. 

Henkens Tom, Vanhaecke Tamara and Rogiers Vera

Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium 

Trichostatin A (TSA) reversibly inhibits histone deacetylase (HDAC) at nanomolar concentrations and has been shown to induce both cell growth arrest and differentiation in several hepatoma cell lines. 
We used primary cultures of rat hepatocytes, to investigate whether TSA is able to maintain their differentiated phenotype in monolayer cultures, undergoing a rapid decrease in hepatic functions following isolation,. Differentiation was assessed by monitoring morphological changes, glutathione S-transferase (GST) activities, 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin O-dealkylase (PROD) activities and albumin secretion throughout culture time. 
Hepatocytes treated with 25�M TSA exhibited longer life span and stay morphologically more differentiated, compared to control cultures, as evidenced by the in vivo-like cuboidal cell shape and the more pronounced presence of bile canaliculi. Also, higher rates of albumin secretion were observed. However, this particular concentration of TSA did not significantly affect phase I and II biotransformation activities.

TSA has been shown a potent inducer of differentiation in several tumor-derived cell lines. However, data on differentiated primary cells are lacking. In primary hepatocytes, the pro-differentiating capacity of TSA is less pronounced. It seems in this particular case, to be restricted to the induction of albumin secretion and morphological differentiation, whereas no effects were observed on xenobiotic biotransformation.

Further studies should be conducted to investigate whether the concentration of TSA and the time of addition to the culture medium play a role. 
 

OC04 - HEPATOCYTES INDUCED FROM PANCREATIC CELLS – A NOVEL APPROACH TO MAKING HEPATOCYTES? 

Chia-Nin Shen, Zo� Burke, Jonathan M.W. Slack and David Tosh

Centre for Regenerative Medicine, Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK 

We have recently produced two models for the transdifferentiation or conversion of pancreatic cells to hepatocytes.  The hepatocytes formed express a wide range of liver proteins including glucose-6-phosphatase, glucokinase, and Phase I (e.g. CYP3A1) and Phase II (e.g. sulphotransferases and UDP-glucuronosyltransferases) metabolising enzymes.  We have determined that the hepatocytes arise as least in part from exocrine cells and that the transcription factor C/EBPbeta is important for the switch from pancreas to liver.  We will present recent evidence on the characterization of the transdifferentiated hepatocytes. 

 

OC05 - CYP INDUCTION IN HUMAN HEPATOCYTES IN MULTIWELL FORMATS - HOW LOW CAN WE GO? 

Andy Sykes, Darren Riddy and Amanda Woodrooffe

Pharmagene Laboratories Ltd, 2A Orchard Road, Royston, Hertfordshire, SG8 5HD 

The objective of this study was to identify the lowest multiwell culture format that allows reproducible induction of CYP activity in hepatocytes freshly isolated from human liver. Hepatocytes, isolated by collagenase digestion, were seeded at 105 viable cells/cm2 on 12, 24 and 48 well collagen coated plates in William’s medium E supplemented with foetal bovine serum (10% v/v), 0.01 g/mL insulin and 0.005 g/mL hydrocortisone (induction medium).  Following cell attachment, the medium was changed to serum free induction medium supplemented with either 1M 3-methylcholanthrene (3-MC), 50M rifampicin, or 1%DMSO (control).  After 72 hours, the cells were washed and incubated with either 50M midazolam or 25M 7-ethoxyresorufin (7-ER) at 37C in serum free induction medium for 2h.  The concentration of 1-OH midazolam was determined by HPLC and the concentration of resorufin was measured using a fluorescent assay.

Treatment with 3-MC resulted in a 3.8, 4.4, and 6.1 fold increase (n=4) in resorufin production in hepatocytes cultured in 12, 24 and 48 well plates respectively, whilst rifampicin produced a 3.9, 5, and 4.5 fold increase (n=2) in 1-OH midazolam production.  In summary, the results from this study suggest there are comparable levels of induction achieved by the standard inducing agents 3-MC and rifampicin when hepatocytes are maintained in culture in 12, 24 and 48 well plates.  This should enable catalytic induction experiments to be performed in smaller well formats and thus provide a more flexible format for such studies.  Future experiments will investigate using alternative culture conditions such as different culture medium or extracellular matrix to optimise the extent of induction. 
 

OC06 - Effect of gender, age, ethnicity and cirrhosis on human liver cytochrome P450 (CYP) enzyme activity and inducibility 

Andrew Parkinson XenoTech, LLC 16825 West 116th Street, Lenexa, KS  66219 

In rats, gender differences in cytochrome P450 (CYP) expression are well documented. Although a recent Japanese regulatory guidance document advocates that CYP enzyme induction studies be conducted in female rats, other guidance documents governing in vitro studies do not specify a preference in gender for human-derived test systems.  Regardless of this, most industry researchers specifically request that human male microsomes or hepatocytes be used for their research or contract studies.  XenoTech has measured CYP activity in almost 200 samples of human liver microsomes, and we have performed induction studies in over 60 preparations of human hepatocytes. We have analyzed this data to examine whether the expression of CYP enzyme activity in liver microsomes and/or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age or ethnicity of the donor. Generally, CYP expression and inducibility are not greatly influenced by gender, age or ethnicity; however, there were insufficient numbers of Asian livers to perform a complete comparison. CYP induction in primary cultures of hepatocytes did not appear to be influenced by the gender or age of the donor, nor did it matter whether the hepatocytes were isolated from histologically normal liver or cirrhotic liver 

 

OC07 - AN ECVAM-SPONSORED PREVALIDATION STUDY ON THE RESPONSE OF PRIMARY HUMAN HEPATOCYTE CULTURES TO MODEL CYP INDUCERS – CRITERIA RETAINED FOR SETTING UP THE INTERLABORATORY HARMONIZED PROTOCOL

Lysiane Richert1 and Edward LeCluyse2

1Biologie Cellulaire, UFR SMP, 4, place Saint-Jacques, 25030 Besan�on and Fondation Transplantation, 5, avenue Moli�re, 67500 Strasbourg, France, 2School of Pharmacy, Division of Drug Delivery and Disposition, University of North Carolina, Chapel Hill, NC, USA

Primary cultures of isolated human hepatocytes have proven to be a valuable model in which to study the inducing potential of drugs on human cytochrome P450 isoenzymes. Because routine evaluation of the inducing potential of a given chemical would be invaluable for human safety assessment, a pre-validation study, sponsored by ECVAM, to evaluate the effects of model compounds belonging to the major classes of known human liver inducers on cultured human hepatocytes using standardized protocols, has been defined. The first part of this talk will focus on the results of our laboratories which have prompted us to identify criteria for setting up the induction studies:

  • human hepatocytes will be isolated either from non-transplantable livers or from surgical resections because in given pre-, intra-, and post-operative conditions, cell yield, viability and function have been shown equivalent (Alexandre et al., submitted),   
  • human hepatocytes will be cultured on collagen-coated dishes, at 125-150 x 105 cells per cm2 and in hormonally-supplemented Hepatocyte Maintenance Medium (HMM) to give optimal response to inducers using a commercially available source of medium for all participating facilities  (LeCluyse et al., 2001),
  • only freshly isolated human hepatocytes will be used, since after crypreservation attachment rate is reduced and a smaller sub-population seems to be selected (Alexandre et al., Cryobiology 2002),
  • A 3-day exposure period with inducers will be utilized because, despite a great variability between control values in hepatocyte cultures from various donors, a treatment-dependent effect could reproducibly be observed within this time frame (Hamilton et al. 2001; LeCluyse et al., 2001; Richert et al., submitted)

In the second part of this talk, the harmonized protocol applied by the 3 partners - Universities of Besan�on/Strasbourg, France - University of North Carolina, USA - University of Leicester, UK-  will be detailed. 
 

OC08 - RECOMBINANT EXPRESSION OF ARYL HYDROCARBON RECEPTOR LIGAND-BINDING DOMAIN 

Tao Jiang, John Fenlon, Ming Qi Fan, Marcos Alcocer and David R. Bell

School of Life & Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD 

The Aryl Hydrocarbon Receptor (AhR) plays an important, if not obligatory, role in the metabolism and toxicity of dioxins, polycyclic aromatic hydrocarbons and several drugs. The AhR is activated to a transcriptionally active heterodimer subsequent to binding of a ligand to the AhR ligand-binding domain (LBD), but little is known about the molecular basis or consequences of this binding.  We have sought to examine the structural basis for the ligand-binding specificity of the AhR. 

A series of deletions were prepared in a minimal functional AhR LBD, covering amino acids 228-416, and the resulting mutants were expressed in reticulocyte lysate to determine the minimal binding domain. The resulting constructs were then expressed in Pichia pastoris, or in Spodoptera frugiperda, using the baculovirus system. High level expression of insoluble AhR LBD was seen in both systems. However, recombinant protein was expressed in yeast in a 10 000g supernatant, but was not present in a cytosol preparation; thus Pichia is very inefficient for expression of soluble AhR LBD. By contrast, high-level expression of soluble AhR LBD was seen in the cytosol fraction of Sf9 cells infected with recombinant baculovirus containing an AhR LBD construct. The optimisation of this system to yield high levels of expression of soluble AhR LBD is described. 

 

OC09 - INDUCTION OF CYP1A, CYP2B, CYP3A AND CYP4A SUBFAMILY FORM mRNAs IN CULTURED PRECISION-CUT RAT LIVER SLICES 

Clive Meredith, Mary P. Scott, Anthony B. Renwick, Roger J. Price and Brian G. Lake 

BIBRA International Ltd., Woodmansterne Road, Carshalton, Surrey, SM5 4DS and Centre for Toxicology, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, GU2 7XH 

Rat liver slices were prepared with a Krumdieck tissue slicer and cultured for 6 and 24 hr in Williams’ medium E supplemented with insulin and dexamethasone (DEX) and also for 24 hr in DEX-free medium. Slices were cultured in control medium or medium containing 10 M -naphthoflavone (BNF), 10 g/ml Aroclor 1254 (ARO), 500 M sodium phenobarbitone (NaPB), 20 M pregnenolone-16-carbonitrile (PCN), 50 M Wy-14,643 (WY) or 50 M methylclofenapate (MCP). Cytochrome P450 (CYP) mRNA induction was determined by real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan�). With the exception of the effect of BNF on CYP1A1 mRNA levels, the induction of all CYP mRNAs studied was greater after 24 than after 6 hr treatment. Generally, the magnitude of induction of CYP mRNA levels was greater after 24 hr in slices cultured in DEX-free medium. The treatment of liver slices with BNF and ARO for 24 hr produced >20 fold increases in CYP1A1 and CYP1A2 mRNA levels. After 24 hr levels of CYP2B1/2 mRNA were increased 9-20 fold by ARO, NaPB, PCN, WY and MCP. PCN also increased CYP3A1 and CYP3A2 mRNA levels and WY and MCP produced a > 150 fold increase in CYP4A1 mRNA levels. These results demonstrate that liver slices can be used to evaluate the in vitro effects of xenobiotics on CYP form mRNA levels. 
 

PC001 - APPLICATION OF MICROARRAY TECHNOLOGY TO EXAMINE PHENOTYPIC CHANGES IN CELL CULTURE: EXAMINATION OF HEPATOCYTE DE-DIFFERENTIATION

 

Toussaint Chevannes-Reeves (1), Lyn Rosenbrier (1), Steve Hood (2), Alan Dickson (1)

(1) 2.205 School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT

(2) MET DMPK, GlaxoSmithKline, Research and Development, Park Rd, Ware, Hertfordshire, SG12 0DP

 

 

Hepatocyte cultures are used as model systems for investigating liver function at the molecular level. However, the isolation and culturing processes of liver cells results in hepatocytes entering a state of dedifferentiation whereby many liver-specific functions are rapidly lost, making this model system unreliable for comparison with the situation in vivo. Work in the laboratory has identified heat shock protein 27 (hsp27) a potential marker of hepatocyte differentiation by differential gene expression analysis. We have gone on to show that hsp27, a stress protein, is expressed in cultured rat hepatocytes after 24 hours of culture and that specific constituents of culture medium (e.g. hydrocortisone and insulin) altered the pattern of its expression. By gaining a better understanding of hsp27 and other associated targets the molecular mechanisms that underlie the dedifferentiation process will be unraveled, therefore enabling prevention of dedifferentiation and improvement of this model system. 

 

PC002 - INFLUENCE OF PRE-, INTRA- AND POST-OPERATIVE PARAMETERS OF DONOR LIVER ON THE OUTCOME OF ISOLATED HUMAN HEPATOCYTES 

Eliane Alexandre1, M�lisa Cahn1, Catherine Viollon-Abadie2, Bruno Heyd2, Georges Mantion2, Daniel Jaeck1 and Lysiane Richert1,2

1Fondation Transplantation, 5 avenue Moli�re, 67200 Strasbourg, France.

2UFR SMP, CHR, 25000 Besan�on, France. 

Isolated human hepatocytes (IHH) represent a unique in vitro system to study drug metabolism and drug-drug interactions. We have explored the suitability of surgical resected liver biopsies, which are available on a more regular basis than non transplanted livers, as a source of viable human hepatocytes. We retrospectively analysed the influence of pre-, intra- and post-operative parameters of donor livers (n=149) on the outcome of IHH yield, viability, attachment rate and function (albumin synthesis and secretion, CYP-dependent activities). To obtain optimal yield and viability: 1) the use of moderate steatotic (> 10 %) or severe cholestatic livers should be avoided, 2) warm ischaemia or clamping time during resection should be less than 30 min, 3) biopsies weighing less than 100 g with cut surfaces being glued, should be used. Attachment rate and function of viable IHH were not affected by those parameters.

In addition, among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of IHH. 
 

PC003 - NANOELECTROSPRAY IONISATION TANDEM MASS SPECTROMETRIC IDENTIFICATION OF CYTOCHROME P450S IN HUMAN LIVER AND HEPATOCYTES

 

S. Nisar1, C. Lane1, A. Wilderspin1, K. Welham1, S. Orr2, S. Kingston2 and L. H. Patterson1

1The School of Pharmacy, 29-39 Brunswick Square, WC1N 1AX and 2The UKHTB, Innovation Centre, Leicester LE1 5XY

 

Levels of cytochrome P450 (CYP) isoforms characterise normal and diseased states. We have applied nanoelectrospray ionisation-tandem mass spectrometry (NSI-MS/MS) as a method of choice for the identification of CYPs from complex protein mixtures.  A method has been developed and optimised using recombinant CYP1A2, 3A4 and 2E1.  NSI-MS/MS coupled with nano-liquid chromatography (nanoLC/MS/MS) provided the sensitivity, specificity and resolution required to identify CYPs from biological media. The method has now been extended to the identification of CYPs from six human livers and one hepatocyte microsomal preparation.  The region between 45 and 68 kDa on SDS-PAGE was cut into five bands, digested with trypsin and resultant peptides were mass analysed.  Amino acid sequences for the peptides were identified using Sequest software. Results from human liver microsomes identified that CYPs 1A2, 2C9, 2C10, 2E1 and 3A4 were present in abundance, while 2A6, 3A5, 2C17, 2D6, 3F2, 4A13 and 4A11 were identified with lower sequence coverage. The hepatocyte preparation contained CYP 2C9, 3A4 and 4A11.  It can be concluded that nanoLC/MS/MS is a reliable method for the simultaneous identification of multiple CYP isoforms at the level of protein expression found in human liver tissue and isolated hepatocytes. The apparently lower CYP complement of human hepatocyte microsomes may be due to the prolonged time required for hepatocyte isolation prior to CYP analysis.

 

 

PC004 - FACTORS AFFECTING THE IN VITRO INTRINSIC CLEARANCE IN HUMAN CRYOPRESERVED HEPATOCYTES

 

Neil Attkins, Nicola Lindsay, Ruth Lock, Daniel Siddle, Kenny Watson, and

Christopher Kohl, Department of Drug Metabolism, PDM, Pfizer Global Research & Development, Ramsgate Rd, Sandwich, Kent, UK

 

Human cryopreserved hepatocytes are potentially the experimental tool of choice for the prediction of hepatic metabolic clearance in the drug discovery phase. Issues are the relatively low initial cell viability of commercially available cryopreserved hepatocytes, the maintenance of their viability during the duration of the incubation, and the low metabolic substrate turn-over rates observed under experimental standard conditions.

The thawing procedure is crucial for the initial cell viability and its maintenance as described earlier by O’Brien et al. (1). Interestingly, improved viability as adjudged by trypan blue exclusion was neither paralleled by faster substrate disappearance rates for diclofenac (acidic), propranolol (basic) and midazolam (neutral at pH 7.4) nor by differences in the activity of phase I or phase II pathways. In contrast, disappearance rates depended on the incubation media used. Drug binding to media constituents such as fetal calf serum, as opposed to non-specific binding to the cells themselves, was shown to be the major determinant of substrate turn-over. The metabolic activity of cryopreserved human hepatocytes towards diclofenac and midazolam was shown to decrease significantly during the course of a three hour incubation (down by 2/3), but it remained constant towards propranolol. This drop in metabolic activity was not accompanied by significant cell death (trypan blue exclusion). The effect of cofactors (NADPH and UDPGA) was shown to be dependent on the presence of non viable and leaky hepatocytes in the incubation mixture. Incorporation of a Percoll centrifugation step, which removes dead and distressed cells, into the cell thawing procedure abolished any cofactor effect on metabolic rates.

Our data show that the utility of cryopreserved human hepatocytes as a model of hepatic metabolic clearance critically depends on the experimental conditions employed. Preliminary results suggest that the different drug metabolising enzymes may lose their activity at different rates during a three hour incubation. Drug substrate binding to the incubation media, as well as cell and substrate concentration, and the incubation time are the major determinants of the in vitro intrinsic clearance observed.

References: Z. Z. O’Brien et al. (2001): Effect of percoll centrifugation on cell viability and enzyme activities of human cryopreserved primary hepatocytes. Drug Met. Rev. 33 (Suppl.1), 191 
 

PC005 - PRE-INCUBATION OF RAT HEPATOCYTES PRIOR TO CRYOPRESERVATION CAN IMPROVE FUNCTION ON THAWING

 

Claire Terry, Sharon C Lehec, Ragai R Mitry, Anil Dhawan and Robin D Hughes

Institute of Liver Studies, Guy’s, King’s and St Thomas’ School of Medicine 

Pre-incubation of hepatocytes prior to cryopreservation, with the aim of boosting levels of intracellular components such as ATP or glutathione, may improve cellular function on thawing.

Rat hepatocytes were isolated using an ex situ perfusion method.  Hepatocytes were pre-incubated with candidate compounds for 1.5 hours at 37�C before cryopreservation (UW solution containing 10% DMSO in a controlled rate freezer).  Control hepatocytes were cryopreserved immediately after isolation.  Hepatocytes were thawed 2 weeks later and viability, protein content, attachment efficiency, MTT, and LDH leakage determined.

Hepatocytes pre-incubated with glucose (0-60 mM) gave significantly higher MTT values than control hepatocytes (5 mM: 49% increase compared to control, p<0.0001; 60mM: 105% increase, p<0.0001).  1 mM ascorbic acid gave significant increases in MTT values (34% increase compared to control, p<0.0001).  -Lipoic acid significantly increased protein content compared to control (0.5 mg/ml: 62% increase, p<0.05; 1 mg/ml: 109% increase, p<0.0001).  The addition of glutathione (0-5 mM) or S-adenosyl-L-methionine (0-5 mg/ml) to the pre-incubation media gave no improvements.

Pre-incubation has the potential to improve metabolic activity and attachment of cryopreserved hepatocytes. 

This work was supported by Merck Sharp and Dohme Ltd (Harlow, UK) 
 

PC006 - VALIDATION OF HUMAN CRYOPRESERVED HEPATOCYTES AS AN IN VITRO MODEL TO STUDY CYTOCHROME P450 INDUCTION.

Dirk Roymans, Dietske Jacobs, Jan Hendrickx, Geert Mannens and Willem Meuldermans

Preclinical Pharmacokinetics, Johnson & Johnson Pharmaceutical Research & Development, Turnhoutsebaan 30, B-2340 Beerse, Belgium.

Current in vitro CYP induction models display major disadvantages. Plateable human cryopreserved hepatocytes (HCH’s) were validated as a model to study cytochrome P450 induction. After plating, cells were allowed to recover for 48h and incubated with 50 �M omeprazole, 25 �M rifampicin, 10 �M dexamethasone or 100 �M phenobarbital for another 48h and compared to non-treated cells. CYP1A2, CYP2C9 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-demethylation, [14C]tolbutamide-methyl-hydroxylation and [14C]testosterone-6-hydroxylation respectively. In addition, mRNA and protein expression was analysed by TaqMan QRT-PCR and immunodetection. HCH activity, protein and mRNA expression was increased 13-, 25-, and 79-fold respectively upon omeprazole treatment. A 2- to 3-fold induction was observed of CYP2C9 mRNA after rifampicin and phenobarbital treatment, while maximally a 17-, 6-, and 24-fold induction was measured of CYP3A4 upon treatment with the same typical inducers. In conclusion, HCH’s may be a good alternative to fresh hepatocytes to study CYP1A and CYP3A induction. 
 

PC007 - STIMULATED TRANSDIFFERENTIATION OF A PANCREATIC CELL LINE TO A HEPATIC PHENOTYPE.

 

Carylyn J. Marek, Gary A. Cameron, Gabrielle M. Hawksworth and Matthew C. Wright

Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Aberdeen, AB25 2ZD, Scotland, UK 

Although the liver is able to regenerate and grow in vivo, hepatocytes rapidly de-differentiate and fail to proliferate to any significant extent in vitro.  This common response of hepatocytes to isolation and culture has limited their utility in for e.g. bio-artificial liver devices. The rat pancreatic cell line AR42J-B13, has therefore been examined for its ability to trans-differentiate into hepatocytes.  Pancreatic AR42J-B13 cells proliferated, were amenable to sub-culture and failed to express liver-specific genes under normal culture conditions.  However, after treating cells with glucocorticoid, proliferation was inhibited.  Over a period of 9 days of continuous glucocorticoid treatment, the cells changed their morphology from a relatively small round phenotype in which cells grew in clusters to an hepatocyte-like morphology (transdifferentiated AR42J-B13 cells).  This change in morphology correlated with an induction of cytochrome P450 (CYP) 2C11 (the major liver-specific cytochrome P450 isoform in rat) such that the levels of expression at 9 days were similar to those expressed in freshly isolated rat hepatocytes.  Other cytochromes P450 were also expressed in hepatic AR42J-B13 cells, including CYP2E1, CYP2A, CYP3A2 and CYP3A1/3A23.  To determine if the expression of cytochrome P450 gene induction in AR42J-B13 cells was functional, cells were treated with the hepatotoxin paracetamol.  This drug requires metabolism by cytochromes P450 for its toxicity.  Paracetamol had little effect on pancreatic AR42J-B13 cells but resulted in significant loss of viability in hepatic AR42J-B13 cells, suggesting that the cytochromes P450 are functional within these cells. This activity was further confirmed by testosterone hydroxylation.

These data indicate that this cell line has an unusual capacity to alter its gene expression profile in response to glucocorticoid.  The mechanism(s) underlying this response are unknown.  However, this work suggests that it may be possible to generate an unlimited supply of hepatocytes in vitro that can be stimulated to differentiate into hepatocytes when required.  A similar human cell line could be essential to the realistic development of a bio-artificial liver for liver bridging support. 
 

 

PC008 - THE ORPHAN NUCLEAR PREGNANE X RECEPTOR (PXR) AND LIVER FIBROSIS 

C.J. Marek*, T. Monaghan$, J.N. Primrose$, M. Koruth+, M. J.P. Arthur$, D.A. Mann$, G.I. Murray*and M.C. Wright*

*Department of Molecular and Cell Biology,University of Aberdeen, Aberdeen, UK; +Aberdeen Royal Infirmary, Aberdeen, UK;  Liver Group, Southampton General Hospital, Southampton, UK. 

Liver fibrosis is a common sequel to liver damage and is characterised by a net accumulation of extracellular matrix proteins within the liver through an imbalance in the deposition and degradation of ECM protein. Hepatic stellate cells (HSCs) play a major role in ECM metabolism, reside in the space of Disse and function in the storage of vitamin A.  In response to liver injury, HSCs proliferate and trans-differentiate to a myofibroblastic - α-smooth muscle actin (α-sma) expressing - phenotype that secretes ECM proteins, proteinases and inhibitors of proteinases.

 

The pregnane X receptor (PXR) is a recently identified orphan nuclear receptor that is transcriptionally activated by pregnanes as well as a number of structurally diverse drugs including rifampicin and metyrapone.  The PXR has been shown to regulate drug- and steroid-inducible expression of cytochrome P450 3A4 , the major expressed cytochrome P450 in human liver responsible for the metabolism of many drugs. 

Treatment of human HSCs in vitro with PXR activators suggests that this receptor may also have an anti-fibrogenic role. To determine whether the PXR is anti-fibrogenic in vivo, rats were administered carbon tetrachloride, either with or without the known rat PXR activator pregnenolone 16-alpha carbonitrile (PCN). These results demonstrate that carbon tetrachloride-induced liver fibrosis is attenuated by PXR activators, however liver damage is unaffected. 
 

PC009 - CRYOPRESERVATION OF PRIMARY PORCINE HEPATOCYTES 

Anne-Dorthe B.Loven, C.Friis, B.Andersen and A.K.Olesen 

The aim of the study was to evaluate the effect of cryopreservation on primary porcine hepatocytes on various metabolic parameters.  

Hepatocytes from four pigs were isolated by way of collagenase and viability was measured using exclusion of trypan blue. The hepatocytes were frozen in freezing boxes in a -80�C freezer for 4 hours and hereafter stored in liquid nitrogen. As controls freshly isolated hepatocytes from the four pigs were plated in 12 well plates. The cryopreserved hepatocytes were thawed in a 37�C water bath and stepwise diluted with buffer while stored on ice. Viability was optimised using Percoll before plating.  

The effect of cryopreservation as well as the effect of a co-culture (rat liver epithelial cells) was evaluated. The parameters were metabolism of paranitrophenol, coumarin and testosterone, and also we looked at carbohydrate metabolism (glucose, lactate and glycogen).  

For the fresh cells viability ranged from 88% to 93%. Initially viabilities for the cryopreserved cells were in the range 60% - 70% and after Percoll treatment the viabilities were increased to 75% - 88%. There seems to be a difference in metabolic capability due to both cryopreservation and co-culture depending on which parameter is examined, but data are still to be evaluated. 

 

PC010 - CULTURED RAT HEPATOCYTES – AN IN VITRO SYSTEM FOR TESTING THE EFFICACY OF RECOMBINANT THERAPEUTIC ANTIBODIES TO PROTECT AGAINST MICROCYSTIN HEPATOTOXICITY 

L.J.Elrick, M.Drevera, J.McElhineyb, L.A.Lawtonb, A.R.Portera and M.C.Wrighta

Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZDa and School of Life Sciences, The Robert Gordon University, St Andrew St, Aberdeen, AB25 1HGb, UK. 

Microcystins are a group of structurally related cyclic peptide hepatotoxins that exert their cytotoxic effect via inhibition of serine-threonine protein phosphatases 1 and 2A.  Exposure to microcystins leads to fulmanent hepatocyte failure, tumour production, acute liver hemorrhage and death.

A na�ve human semi-synthetic phage display library was used to isolate recombinant fragments against microcystin-LR.  Selected antibody scFv genes were cloned into an appropriate expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA).  Isolated single-chain antibodies (scab) were capable of detecting microcystin-LR at levels below the World Health Organisation recommended limit in drinking water (1 g/litre-1).

Treatment of freshly isolated rat hepatocytes with increasing concentrations of microcystin-LR induced apoptosis with decreasing time.  A concentration of 100 nM was sufficient to promote apoptotic body formation, confirmed by dapi staining and fluorescence microscopy, within 6 hours.  Incubation of hepatocytes with an anti-microcystin-LR scab, in the presence or 100 nM microcystin-LR, was necessary and sufficient to prevent apoptotic body formation and cell death.

These preliminary results demonstrate a novel therapeutic application of recombinant antibody technology to protect and prevent liver insult in response to environmental microcystin exposure. 
 

PC011 - EXPERIENCE OF LARGE SCALE ISOLATION OF HUMAN HEPATOCYTES AT KING’S COLLEGE HOSPITAL 

R. R. Mitry, R. D. Hughes, S. Lehec, C. Terry, G. Mieli-Vergani, M. Rela, N.D. Heaton, R. Girlanda, P. Muiesan, A. Dhawan.

Institute of Liver Studies, King’s College Hospital, London, UK. 

Hepatocyte transplantation is being developed at King’s College Hospital for treatment of paediatric liver patients with inborn-errors of metabolism, and acute liver failure. We have established large scale hepatocyte isolation using a collagenase perfusion technique with purification by washing/slow speed centrifugation according to Strom et al. (1998) with some modifications.  Hepatocytes were isolated from 42 donor tissues (21 right lobe; 9 left lobe; 10 whole livers; 2 segment IV). The mean cell number obtained was 2.9x109 � SD0.6x109 with a mean cell viability of 64 � SD22%. These tissues included 27 unused donor lobes/segments from split or reduced grafts (cell viability 74 � SD19%), and 15 tissues (10 being steatotic) that were rejected for clinical transplantation (cell viability 47 � SD17%; p<0.001).  Livers from non-heart beating donors are being evaluated as an alternative source of hepatocytes and in 13 livers (11 rejected for transplantation), the mean cell viability was 45 � SD19%.

Eleven of these procedures were carried out under strict GMP sterile conditions in our purpose-built King’s Cell Isolation Unit. Clinical application of hepatocyte transplantation has recently started.  Five of the clinical grade cell preparations (fresh and cryopreserved) were used for hepatocyte transplantation in two children with genetic liver disease. 

 

PC012 - CRYOPRESERVATION OF HEPATOCYTES: THE MONOLAYER APPROACH 

David Stevenson, Helen Grant, Elizabeth Goldie, Gail Connel & Caroline Morgan

Bioengineering Unit, Strathclyde University, 106 Rottenrow, Glasgow G4 0NW 

The ability to cryopreserve hepatocytes would be useful both to the pharmaceutical industry and for bioartificial liver support systems.  Unfortunately, suspension cryopreservation protocols typically result in low attachment efficiencies of cells upon thawing.  To circumvent this problem, we have frozen rat hepatocytes as monolayers on collagen substrates, and attempted to optimise this cryopreservation protocol.     

A variety of parameters were measured in non-frozen and post-thaw frozen monolayer cultures, including viability, total protein and  intracellular reduced glutathione (GSH) concentration, kaempherol glucuronidation, and testosterone hydroxylation.  The effect of altering cryopreservation media composition (% of foetal calf serum varying between 0-90%) or freezing (0.4-3.8�C/min) and thawing rates (26-128�C/min) on these parameters was investigated.   

Under optimal conditions, post thaw cryopreserved cells maintained 72�4% viability, 65�4% total protein, 46�8% GSH, 48�8% kaempherol glucuronidation, and 16�11% testosterone hydroxylation of their corresponding non-frozen controls (mean �SEM, n=3).  Cryopreservation of hepatocyte monolayers as opposed to suspensions results in a more representative population of cells, with high viability, function, and recovery rates.      
 

PC013- Human Hepatocyte tissue banking- Consumer views and future challenges 

Tom Lloyd, Samantha Orr1, Jacki Trafford1, Carol Granger, Ashley Dennison

Department of Surgery, Leicester General Hospital, Leicester.  LE5 4PW

1UK Human Tissue Bank, Innovation Centre, De Montfort University, Leicester.

 

Introduction

The UK human tissue bank routinely isolates human hepatocytes with acceptable and consistent cell viability. A major focus of the UKHTB strategy is to supply freshly isolated human hepatocytes. Analysis reveals that only 42% of these isolated cells are being utilised fresh. In efforts to maximise this precious resource a telephone survey was conducted of researcher requirements.

 

Method

Questions focussed on cell usage, quality of hepatocytes on arrival, logistics, future plans and perceived problems of work utilising human hepatocytes. Opinions on the value of cryopreserved and plated hepatocytes were also sought.  

Results

Twelve researchers were contacted. Problems cited included unsociable hours, personnel and health and safety issues. No significant problems were reported with the notification and transport of the cells, although last minute cancellations caused frustration. Cellular viability and cellular function was generally good.

 

Conclusion

Easily accessible, good quality human hepatocytes are now routinely available to researchers in the UK. The availability at unsociable hours remains a problem to gain maximum usage of the cells isolated. Current efforts being implemented include research into cryopreservation, increased transport of pre-plated hepatocytes and overnight cold storage of fresh tissue. Other future plans to improve logistics include increasing the number of livers donated and isolation centres. 

 

PC014 - THE INFLUENCE OF PATIENT AND ISOLATION FACTORS ON SUBSEQUENT YIELD, VIABILITY AND FUNCTION OF HUMAN HEPATOCYTES 

Tom Lloyd, Joy Wilkinson, Rakhee Patel1, Gerard Crees1, Seema Chavda1, Samantha Orr1, Ashley Dennison.

Dept of Surgery, Leicester General Hospital, Leicester.  LE5 4PW

1UK Human tissue Bank, Innovation Centre, De Montfort University 

Introduction

The UK Human tissue bank undertakes large scale isolation of human hepatocytes from donations of surgically resected liver tissue and non-transplantable livers from brain stem dead multi organ donors (MOD). This study seeks to identify the influence of patient, operative, isolation and/or transport prior to isolation on yield, viability and function of the isolated human hepatocytes.  

Method

Patient and hepatocyte isolation details have been recorded since October 2001 to the present day. A multi variant analysis has been undertaken to ascertain the influence of such factors. 

Results

To date hepatocytes have been isolated from 65 surgically resected livers, and 5 multi organ donor livers. Early analysis has found that surgically resected tissue gave superior yields and cellular viability when compared to non-transplantable livers. Journey times of up to 5 hours prior to isolation from resected livers has no significant effect on the quality of the hepatocytes isolated.  

Conclusions

This study includes the analysis of multiple patient and procedural factors that may influence subsequent yield, viability and function of isolated hepatocytes. It is hoped that this will lead to a better understanding of the isolation process as well as predicting the suitability of donated liver for hepatocyte isolation. 
 

 

PC015 - STUDIES INTO THE OPTIMISATION OF HUMAN HEPATOCYTE CRYOPRESERVATION

 

Tom Lloyd, Samantha Orr, Ashley Dennison

 

Introduction

To optimise the effective banking of human hepatocytes, studies were undertaken to develop a protocol for cryopreservation.

 

Method

Our studies comparing the controlled rate freezer with the Nalgene isopranolol devicehave revealed the latter to be the most effective controlled temperature decrease method. The effect of four different human hepatocyte cellular densities (2.5x106, 5x 106, 1x107, 2x107/ml), and the influence of culturing the cells in a spinner flask for 1hour and 16hours prior to cryopreservation and subsequent culture has been evaluated. Cell recovery and viability (measured using trypan blue exclusion), attachment efficiency, bilirubin conjugation, LDH leakage and lignocaine metabolism (CYP 3A4) were measured after 2 and 5 days of culture. These studies were also run in parallel using porcine hepatocytes.

 

Results

Our data (n=16) reveals the optimal concentration for number of hepatocytes recovered to be 5x106/ml. Functional parameters reveal no significant difference. Pre-culture (1hr and 16hr) results in recovery of 60% viable cells with superior attachment efficiency(n=7). Full functional statistical analysis will be presented.

 

Conclusion

Early data suggests that cryopreserved cells function in a similar fashion to fresh cells, at least in the short term. The optimisation of a cryopreservation protocol is an important step in maximising the human liver tissue donated to UKHTB. 
 

PC016 - EXPRESSION OF CYP3A23, CYP3A2, CYP3A9 AND PXR IN RESPONSE TO DEXAMETHASONE INDUCTION IN VITRO 

S.J. Lucas1, P.R. Murdock*, A.D. Ayrton*, R.J. Chenery* and G.M. Hawksworth

1Depts. Medicine and Therapeutics and Biomedical Sciences, University of Aberdeen.

2GlaxoSmithKline, Stevenage and  The Frythe, Welwyn, Herts.

Induction of cytochrome P450 (CYP) by drugs can significantly alter pharmacological and toxicological responses in both experimental animals and in man.  Induction is traditionally determined by measuring immunoreactive protein content by Western blotting, enzyme activity levels and, more recently, by competitive RT- PCR.  However, these techniques are both labour intensive and time consuming. We have used real-time quantitative RT-PCR (TaqMan PCR) to measure the expression of rat CYP3A isozymes and PXR in hepatocyte cultures. 

Probe and primer sets were developed for rat CYP3A23, CYP3A2 and CYP3A9, and for PXR. Total RNA was isolated from primary cultures of adult male rat hepatocytes following 24-hour incubation with dexamethasone (DEX).  CYP3A23 was maximally induced 329-fold, compared with 2 to 3-fold changes in CYP3A2, CYP3A9 and PXR mRNA.  CYP3A23 showed a biphasic response to DEX treatment.  Low amplitude induction (approx 7-fold) occurred at nanomolar (50-100 nM) concentrations and high amplitude induction (approx 300-fold) at micromolar (0.5-3 M) concentrations.  PXR mRNA expression increased 3-fold up to 100 nM DEX and then decreased at micromolar concentrations.  These results are in line with the findings of Pascussi et al., (2000) who first demonstrated a biphasic response of CYP3A4 to DEX in cultured human hepatocytes.  However, we have observed that, in rat hepatocytes, the low and high components of DEX-mediated induction occur at lower concentrations and cause greater fold increases in CYP3A expression.  This is in agreement with the finding that DEX is a better activator of rat PXR compared with human (Jones et al., 2000).  In conclusion, TaqMan PCR provides an extremely sensitive method for mechanistic analyses of cytochrome P450 inducers acting at the level of gene transcription. 

 

PC017 - DIFFERENTIAL EFFECTS OF CANNABIDIOL (CBD) AND 9-TETRAHYDROCANNABINOL (THC) ON INDUCTION OF RAT CYTOCHROME P450S (CYPS) FOLLOWING IN VIVO ADMINISTRATION. 

1McArdle, K. E., 1Pertwee, R. P., 2Guy, G. W., 2 Whittle, B. A., 1 Hawksworth, G. M.

1Departments of Medicine and Therapeutics and Biomedical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, 2 GW Pharmaceuticals, Science Park, Porton Down, Salisbury, Wiltshire, UK SP4 0JO.

k.mcardle@abdn.ac.uk 

The co-administration of two or more cannabinoids (e.g. CBD and THC) presents the potential for drug-drug interactions, which could lead to altered pharmacological and therapeutic effects.  CBD has been shown to differentially inhibit the oxidative metabolism of THC in human hepatic microsomes (McArdle et al., 2001, Jaeger et al., 1996).  The aim of this study was to evaluate the ability of CBD and THC to induce CYPs in vivo in rat. 

Male Sprague Dawley rats (n = 4) were administered intraperitoneally (i.p) either CBD or THC (15, 50, 150mg/kg in corn oil (CN)) once daily for 3 days.  Dexamethasone (DEX 150mg/kg in corn oil), Sodium phenobarbitone (PB 80mg/ kg in saline (SAL)) or -naphthoflavone

(BN 40mg/kg in corn oil) were also administered i.p once daily for 3 days, as positive controls.  The livers were removed on day 4 and hepatic microsomes were prepared.  The extent of CYP induction was measured by Western immunoblotting using 1g protein.

 

Figure 1 Induction of rat cytochrome P450 proteins by CBD

 

There was no induction of CYP1A1 with CBD treatment.  However, CYP1A2 was induced in a dose-dependent manner (maximum at 150mg/kg) (Figure 1).  This suggests that CBD induction

of CYP1A2 may be regulated via an AhR-independent pathway.  CBD also induced rat CYP3A23 and CYP2B1/2 with the greatest increase at 150mg/kg (Figure 1).  In contrast, THC failed to induce any of the CYPs assessed, despite being structurally related to CBD.  Although the inducing effects of CBD on human CYPs have not yet been determined, these results highlight the potential for CBD to interact with other co-administered drugs. 

McArdle K, Mackie P, Pertwee R, Guy G, Whittle B and Hawksworth G (2001) Toxicology  
168: 133 – 134.

Jaeger W, Benet L and Bornheim L (1996) Xenobiotica 26: 275 – 284. 
 

 

PC018 - A POST-TRANSCRIPTIONAL MECHANISM IS RESPONSBILE FOR SUPPRESSED CYP2B INDUCTION IN FEMALE WISTAR KYOTO RATS 

Cliff Rowe1, Andrew Ayrton2, Richard J. Chenery2, Steve R. Hood2 Gabrielle M. Hawksworth*1

1Depts. Medicine and Therapeutics and Biomedical Sciences, University of Aberdeen, AB25 2ZD.

2DMPK Dept., GlaxoSmithKline, Park Road, Ware, Herts SG12 ODP. 

Cytochrome P450 2B1 (CYP2B1) is not expressed in normal rat liver but is induced by phenobarbitone (PB), which up-regulates the gene via the constitutively active receptor. The response to PB is suppressed in female Wistar Kyoto (WKY) rats which have no detectable CYP2B1 protein 24 hours after PB administration. To investigate the basis of this suppression, female WKY and Wistar rats were administered a single dose of PB (100mg/kg) or saline (2ml/kg) (n=4 per group) and killed after 24 hours. CYP2B1 mRNA in the liver was quantified by Taqman� real time PCR. CYP2B protein in liver microsomes was detected by Western blotting.

Control rat livers from both strains had no detectable CYP2B1 mRNA. WKY rats averaged 1918 copies of CYP2B1 mRNA/ng total liver RNA (range: 705-2506) and Wistar rats averaged 1182 copies (range: 862-1597) after PB induction.  PB treated WKY rats did not translate CYP2B protein, but protein was detectable in liver microsomes made from PB treated female Wistar rats. PB also induced levels of CYP3A23 mRNA in both strains but, in contrast to Wistar rats, WKY rats failed to translate this to protein.

The suppression was post-transcriptional and was not evident for isosafrole-mediated induction of CYP1A or dexamethasone-mediated induction of CYP3A. 
 

PC019 - CELLULAR DAMAGE TO HUMAN HEPATOCYTES THROUGH REPREATED APPLICATION OF 5-AMINOLEVULINIC ACID 

T.S. Weiss, S. Pahernik, I. Scheruebl, K.-W. Jauch, W.E. Thasler

Center for Livercell Research, University of Regensburg Hospital, F.-J.-S.-Allee 11, 93053 Regensburg, Germany 

Background/Aims:  5-aminolevulinic acid (ALA), a precursor of porphyrins is used for photodynamic diagnosis and therapy within topical or systemic applications. A potential toxic effect on the human liver is of major interest and therefore we investigated the impact of a repeated application of ALA without illumination on cultures of human hepatocytes.

Methods: After ALA treatment of hepatocytes in vitro the porphyrin synthesis, albumin secretion, liver-specific enzyme release, and malondialdehyde levels were determined. In order to reduce levels of reactive oxygen substances, mannitol and the antioxidant enzymes superoxide dismutase and catalase were supplemented.

Results:  Porphyrin biosynthesis by human hepatocytes in vitro was repeatedly stimulated by ALA (0.001 - 1.0 mM), which was indicated by an accumulation of protoporphyrin IX. A repetitive treatment (up to 4 times) of hepatocytes with ALA resulted in an impairment of the hepatic function and viability, depending on the ALA concentration (0.1 - 1.0 mM) and frequency of application (2-3 times). This was also accompanied by increased malondialdehyde levels indicating enhanced lipid peroxidation. Only superoxide dismutase was able to reduce cellular damage and prevent specific function.

Conclusions:  Repeated, not single, ALA treatment without illumination may cause deleterious effects to the liver, which are mediated by oxygen radicals and inhibited by an antioxidant. 
 
 

 

PC020 - ENGINEERING FUNCTIONAL LIVER TISSUE IN VITRO: HEPATOCYTE/STELLATE CELL SPHEROIDS 

R. Bhandari1, L. Riccalton-Banks1, J Fry2, A. Bennett2, K. Shakesheff1.

1School of Pharmaceutical Sciences, 2School of Biomedical Sciences, University of Nottingham 

The development of a liver tissue model capable of mimicking in vivo functionality with respect to drug metabolism and toxicology poses a major challenge. Removed from their natural cell-cell interactions and environment, hepatocytes in culture begin to lose CYP450 activity within hours and only remain viable for a few days. In vivo, hepatic stellate cells appear to form intimate physical contacts with hepatocytes. Incorporation of these interactions in the design of in vitro functional liver-like tissues should preserve at least some of the signals and cues hepatocytes encounter in vivo. Rapid spheroid formation using primary isolated hepatocytes and hepatic stellate cells on a biodegradable poly (lactic acid) substratum is described (International patent application No. PCT/GB01/05566). The 3-dimensional co-cultured spheroids maintained viability and

hepatocyte-specfic functionality (EROD activity and albumin secretion) for over 7 weeks (Tissue Engineering 9 (3) 2003). Initial results will be presented of studies underway to elucidate the possible mode of regulation of specific CYP450 isoforms and components of the cell-cell interactions involved in enhancing viability and preserving hepatocyte-specific functionality.


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