Fresh Hepatocytes Research Articles

Prediction of metabolic clearance using fresh human hepatocytes: Comparison with cryopreserved hepatocytes and hepatic microsomes for five benzodiazepines

1. Predictions of in vivo intrinsic clearance from cryopreserved human hepatocytes may be systematically low. In the current study, the metabolite kinetics of a series of CYP3A4 substrates (benzodiazepines) in fresh human hepatocytes from five donors, via a major UK supplier, were investigated and compared with those previously reported (by the authors’ laboratory) for cryopreserved human hepatocytes and hepatic microsomes.2. A high incidence of autoactivation (up to tenfold) and heteroactivation (by testosterone, up to 14-fold) among the major pathways was observed. CYP capacity (Vmax) was marginally lower and ‘affinity’ constants (KM, S50) were marginally greater compared with cryopreserved hepatocytes.3. Average intrinsic clearance (based on maximal clearance, CLmax) was sevenfold lower than in cryopreserved hepatocytes (reflecting sensitivity of intrinsic clearance estimation in vitro to mechanistic parameter values, particularly those involving atypical kinetics), but scaled intrinsic clearances for fresh (and cryopreserved) hepatocytes were within the range previously determined in hepatic microsomes.4. There was no evidence from this series of studies that fresh hepatocytes provide quantitatively improved estimates of intrinsic clearance over cryopreserved hepatocytes.

A direct effect of erythropoietin on hepcidin expression involving EPOR signaling

Background/Aims: Hepcidin is the main iron regulatory hormone, controlling the systemic absorption and remobilization of iron from stores. In vivo studies showed that hepcidin is down‐regulated by erythropoiesis, anemia and hypoxia, which conforms to the need for iron input for erythrocyte production. The hormone erythropoietin (EPO) is the primary signal triggering erythropoiesis in anemia and hypoxia. Therefore, a direct involvement of erythropoietin in hepcidin regulation can be hypothesized. The aim of this study was to test the effect of EPO on hepcidin expression in vitro and to begin characterization of the mechanisms involved.Methods: Fresh mouse hepatocytes and the HepG2 cell line were supplemented with physiological and supra‐physiological doses of EPO. Blocking of EPOR was performed with a specific antibody. Quantification of hepcidin and C/EBPA mRNA was performed by quantitative Real‐Time PCR. C/EBPA protein levels were quantified by Western blot hybridization.Results: EPO regulates hepcidin expression in HepG2 cells and in mouse hepatocytes, in a dose‐dependent manner. This effect is mediated through EPOR signaling, since hepcidin expression is restored by EPOR‐blocking. A pattern of expression similar to hepcidin after EPO and α‐EPOR treatments was observed for CEBPA at mRNA and protein levels, suggesting the involvement of this transcription factor in the regulation of hepcidin by EPO.

Gene expression predicts differential capecitabine metabolism, impacting on both pharmacokinetics and antitumour activity

Capecitabine is converted into 5′-deoxy-5-fluorocytidine (5′DFCR), 5′-deoxy-5-fluorouridine (5′DFUR) and 5-fluorouracil (5-FU) by CES1 and 2, CDD, and TP, in both liver and tumour. 5-FU is catabolised by DPD. Gene expression analysis of these enzymes was undertaken in fresh human hepatocytes, mouse liver, colorectal cancer cell lines and xenografts. Cell lines with low CDD expression (<1.5) had 5′DFCR/5′DFUR cytotoxicity ratios >2 and cell lines with TP/DPD < 0.6 had 5′DFUR IC 50 > 50 μM (SRB assay). A pharmacokinetic/pharmacodynamic study in nude mice bearing HCT 116 xenografts and treated with capecitabine by oral gavage assessed pharmacokinetic, gene expression and antitumour activity. Low liver CDD correlated with high 5′DFCR plasma concentrations in mice. CDD expression was ∼100-fold higher in fresh human hepatocytes than mouse liver, explaining the higher plasma 5′DFUR concentrations reported previously in humans. Tumour 5-FU concentration correlated with TP/DPD and with tumour response. These studies identify the potential utility of gene expression analysis and drug monitoring in tumour in patients.

Investigations Into the Drug-Drug Interaction Potential of Tapentadol in Human Liver Microsomes and Fresh Human Hepatocytes

The new analgesic tapentadol was evaluated for induction and inhibition of several cytochrome P450 enzymes in vitro, and protein binding was assessed. It was concluded that no clinically relevant drug-drug interactions are likely to occur through either mechanism.

Native Umbilical Cord Matrix Stem Cells Express Hepatic Markers and Differentiate Into Hepatocyte-like Cells

Umbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage. We isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins. In vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated. We conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies.

Prediction of drug-induced liver injury in humans by using in vitro methods: The case of ximelagatran

Objective: To investigate the possible mechanisms underlying the liver enzyme elevations seen during clinical studies of long-term treatment (>35 days) with ximelagatran, and investigate the usefulness of pre-clinical in vitro systems to predict drug-induced liver effects.Methods: Ximelagatran and its metabolites were tested for effects on cell viability, mitochondrial function, formation of reactive metabolites and reactive oxygen species, protein binding, and induction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) gene expression or nuclear orphan receptors. Experimental systems included fresh and cryopreserved hepatocytes, human hepatoma cell lines (HepG2 and HuH-7) and subcellular human liver fractions.Results: Loss of cell viability was only seen in HepG2 cells at ximelagatran concentrations ⩾100μM and in cryopreserved human hepatocytes at ⩾300μM, while HuH-7 cells were not affected by 24h exposure at up to 300μM ximelagatran. Calcium homeostasis was not affected in HepG2 cells exposed to ximelagatran up to 300μM for 15min. There was no evidence for the formation of reactive metabolites when cell systems were exposed to ximelagatran. ALT and AST expression in human hepatoma cell lines were also unchanged by ximelagatran. Mitochondrial functions such as respiration, opening of the transition pore, mitochondrial membrane depolarization and β-oxidation were not affected by ximelagatran or its metabolites.Conclusion: Ximelagatran at concentrations considerably higher than that found in plasma following therapeutic dosing had little or no effect on cellular functions studied in vitro. The in vitro studies therefore did not elucidate the mechanism by which ximelagatran induces liver effects in humans, possibly because of limitations in the experimental systems not reflecting characteristics of the human hepatocyte, restricted exposure time, or because the primary mechanism for the observed clinical liver effects is not on the parenchymal liver cell.

Cytosolic LC3 Ratio as a Sensitive Index of Macroautophagy in Isolated Rat Hepatocytes and H4-II-E Cells

Macroautophagy, an intracellular bulk degradation process in eukaryotes, is sensitive to nutrient supply and deprivation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an indispensable role in macroautophagy formation and is a suitable marker for this process. Through analysis of the subcellular distribution of LC3, we determined that the cytosolic fraction contained not only a precursor form (LC3-I), but also an apparent active form (LC3-IIs). Both cytosolic LC3-I and LC3-IIs were more responsive to amino acids than those of total homogenate. Moreover, changes in the LC3-IIs/I ratio reflected those in the total proteolytic flux remarkably in both fresh rat hepatocytes and H4-II-E cell lines. Thus, in addition to a sensitive index of macroautophagy, calculating the cytosolic LC3 ratio became an easy and quick quantitative method for monitoring its regulation in hepatocytes and H4-II-E cells.

Method for rapid metabolite profiling of drug candidates in fresh hepatocytes using liquid chromatography coupled with a hybrid quadrupole linear ion trap

Rapid information on metabolic profiling is required to evaluate the structural liabilities of drug candidates in early drug discovery. In this study, a sensitive and rapid semi-quantitative method was developed to simultaneously monitor the drug candidate and metabolites as well as collect tandem mass (MS/MS) spectra for subsequent metabolite identification. The simultaneous semi-quantitation and identification of metabolites in fresh hepatocytes is achieved using high-performance liquid chromatography (HPLC) coupled with a hybrid quadrupole linear ion trap. The survey experiment consists of monitoring multiple-reaction monitoring (MRM) transitions for the internal standard, the parent, and 48 MRM transitions designed to cover the most common phase I and II biotransformations. An information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold. Three biotransformations of a lead compound have been identified through enhanced product ion scans and the respective MRM transitions of those metabolites were selected for semi-quantitation. Parent disappearance and formation of the metabolites as a function of incubation time in five different species were monitored by their respective MRM responses. The method provides the necessary sensitivity to detect minor metabolites in a relevant therapeutic concentration range. Enzymatic turnover of the parent and the metabolites in different species are revealed based on the different initial concentrations of the parent. This methodology integrates the parent disappearance, metabolite identification, and the formation of the metabolites along the time course using a single rapid LC/MS/MS analysis. This method can be used as a complementary tool to the conventional method of metabolic profiling. It provides a rapid and sensitive initial profile of the metabolism of potential structural series at the lead selection stage. The method can also be incorporated into the overall metabolite profiling scheme to evaluate the drug candidates in drug discovery.

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.

Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and BMP2/4

AbstractThe peptide hormone hepcidin is the principal regulator of systemic iron homeostasis. We examined the pathway by which iron stimulates the production of hepcidin. In humans who ingested 65 mg of iron, the increase in transferrin saturation preceded by hours the increase in urinary hepcidin excretion. Increases in urinary hepcidin concentrations were proportional to the increment in transferrin saturation. Paradoxically, in previous studies in primary hepatocytes and cell lines, hepcidin response to iron or iron transferrin was not observed. We now report that freshly isolated murine primary hepatocytes responded to holotransferrin but not apotransferrin by increasing hepcidin mRNA. Hepcidin increase was not due to contamination of the transferrin preparations by endotoxin, a potent pathologic stimulus of hepcidin synthesis. Using this culture system, we showed that holotransferrin concentrations regulate hepcidin mRNA concentrations through a hemojuvelin/BMP2/4–dependent pathway. Although BMP9 is known to be expressed in the liver and potently increased the basal concentrations of hepcidin mRNA, it did not interact with hemojuvelin, and interference with its signaling pathway did not affect iron regulation. Fresh primary hepatocytes constitute a sufficient system for the regulation of hepcidin by physiologic iron stimuli and will greatly facilitate studies of major disorders of iron homeostasis.

The evaluation of an in vitro fresh suspension hepatocyte model in the investigation of drug-induced hepatotoxicity

The evaluation of an in vitro fresh suspension hepatocyte model in the investigation of drug-induced hepatotoxicity

Cryopreservation-Induced Nonattachment of Human Hepatocytes: Role of Adhesion Molecules

Good quality cryopreserved human hepatocytes are becoming an important source for clinical hepatocyte transplantation. However, the process of cryopreservation leads to both structural and functional impairment of hepatocytes. The aim of this study was to investigate the mechanisms of cryopreservation-induced nonattachment in human hepatocytes. Hepatocytes were cryopreserved after isolation from unused donor liver tissue. Cell attachment to collagen-coated plates was measured. A cDNA gene array system for 96 cell adhesion-related molecules was used to determine mRNA expression in fresh and cryopreserved hepatocytes. Two cell adhesion molecule proteins were investigated further: beta1-integrin, a cell-matrix adhesion molecule, and E-cadherin, a cell-cell adhesion molecule. Attachment efficiency was significantly decreased after cryopreservation of human hepatocytes. Twenty-two genes were downregulated after cryopreservation including integrins, cadherins, catenins, and matrix metalloproteinases (MMPs). Beta1-Integrin gene and protein expression were significantly decreased in cultured cryopreserved hepatocytes compared to fresh hepatocytes. There was a significant correlation between loss of beta1-integrin and attachment in cryopreserved cells. Degradation of E-cadherin was increased in cryopreserved hepatocytes. The process of cryopreservation leads to downregulation of cell adhesion molecules at the gene and the cellular level. New cryopreservation protocols are needed to prevent these effects on cell attachment.

Impact of Physicochemical and Structural Properties on the Pharmacokinetics of a Series of α1L-Adrenoceptor Antagonists

A rational drug discovery process was initiated to design a potent and prostate-selective alpha1(L)-adrenoceptor antagonist with pharmacokinetic properties suitable for once a day administration after oral dosing, for the treatment of benign prostatic hyperplasia. Two series of compounds based on a quinoline or quinazoline template were identified with appropriate pharmacology. A series of high molecular weight cations with high hydrogen-bonding potential had extensive in vivo clearance, despite demonstrating metabolic stability. Studies in the isolated perfused rat liver and fresh rat hepatocytes indicated that active transport protein-mediated hepatobiliary elimination is an efficient clearance process for these compounds. A reduction in molecular weight and hydrogen-bonding potential resulted in a second series of compounds with in vivo hepatic clearance predictable from in vitro metabolic clearance. Initially, lipophilicity was reduced within this second series to reduce metabolic clearance and increase elimination half-life. However, this strategy also resulted in a concomitant reduction in volume of distribution and a negligible effect on prolonging half-life. An alternative strategy was to increase the intrinsic metabolic stability of the molecule by careful structural modifications while maintaining lipophilicity. Replacement of the metabolically vulnerable morpholine side chain resulted in identification of UK-338,003, (N-[2-(4-amino-6,7-dimethoxy-5-pyridin-2-yl-quinazolin-2-yl)-1,2,3,4-tetrahydro-isoquinolin-5-yl]-methanesulfonamide), which fulfilled the objectives of the discovery program with suitable pharmacology (human prostate alpha1(L) pA(2) of 9.2 with 25-fold selectivity over rat aorta alpha1(D)) and sufficiently long elimination half-life in human volunteers (11-17 h) for once a day administration.

Assessment of Cryopreserved Hepatocytes as an Alternative to Fresh Hepatocytes forComparative InterspeciesMetabolism Studieswith Suitable Acceptance Criteria

Fresh hepatocytes have been the choice for interspecies comparative drug metabolism studies. Cryopreserved hepatocytes represent a readily available alternative when combined with acceptance limits based on the metabolic turnover of 7-ethoxycoumarin. Results for the ten NCEs examined show that the metabolites formed were strongly correlated in fresh and cryopreserved hepatocytes.

Same-sample analysis of ethoxyresorufin- O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes

Inducibility of the cytochrome P4501A4 (CYP1A4) enzyme, measured as ethoxyresorufin- O-deethylase (EROD) activity, has been used as a biomarker for sensitivity to the effects of dioxin-like compounds in avian species. Here, we present a quantitative reverse transcriptase-PCR (Q-PCR) method for assessing this biomarker response at the level of messenger RNA (mRNA) expression. The method was validated for use in fresh samples as well as samples that have been analyzed for EROD activity previously. Concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on CYP1A4 and CYP1A5 mRNA abundance were detected in fresh and post-EROD hepatocyte cultures. Although the quality of the RNA obtained from post-EROD samples was low, quantification of the CYP1A mRNA response to TCDD was not compromised. Several benefits of evaluating CYP1A mRNA expression in addition to EROD activity were noted. The CYP1A mRNA bioassay may provide more accurate estimates for the potency of environmental mixtures of contaminants and has a very low detection limit. When working with hepatocytes cultured from wild or endangered species, our approach can help to circumvent the problem of small sample size by maximizing the amount of data obtained from each sample.

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 × 10 4 cells/cm 2 and 2.1 × 10 4 cells/cm 2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 × 10 4 cells/cm 2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response ( 3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain α1-receptor function.

Use of Cryopreserved Human Hepatocytes in Sandwich Culture to Measure Hepatobiliary Transport

Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 x 10(6) cells/well in 500 microl, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 microl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 microl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.

Analysis of the Effects of Cryopreservation on Rat Hepatocytes Using SELDI-TOF Mass Spectrometry

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.

Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes

Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent.

Rat hepatocyte engraftment in severe combined immunodeficient × beige mice using mouse‐specific anti‐fas antibody

Hepatocyte transplantation holds promise as a treatment for acute and chronic liver failure; however, robust model systems needed to study xenogeneic hepatocyte transfer are lacking. Severe combined immunodeficient x beige (SCID/bg) hybrid mice readily accept foreign tissue. Repopulation of C.B-17 SCID/bg mouse liver with rat hepatocytes was studied following induction of mouse hepatocyte apoptosis using an anti-mouse agonistic fas monoclonal antibody (Jo2 mAb) that does not engage xenogeneic fas. SCID/bg mice were transplanted with 1 x 10(6) fresh adult rat hepatocytes intrasplenically and treated with various doses, routes and frequencies of Jo2 mAb. Rat cell repopulation was characterized by quantitative immunofluorescent antibody (q-IFA) staining specific for rat dipeptidyl peptidase type IV (DPP-IV) and leucine amino peptidase, amplification of rat genomic DNA using polymerase chain reaction and histopathological and serum biochemistry analyses. Analysis of liver sections from mice treated twice weekly for 12 weeks with 0.4 mg/kg Jo2 mAb intraperitoneally consistently demonstrated >50% rat hepatocytes in the parenchymal mass by q-IFA. Rat hepatocyte engraftment protected mice from Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. Serum liver enzyme levels did not increase in Jo2 mAb-treated mice that were highly engrafted with rat hepatocytes, in contrast to matched non-engrafted mice. At 12 weeks post-engraftment, minimal fibrosis and inflammation were apparent and liver architecture had returned to near normal. Jo2 mAb did not induce histopathological abnormalities in other tissues known to express fas antigen (i.e. heart, lung). This novel model represents a simple and robust system of xenogeneic hepatocyte transplantation that could be applied to studies of liver biology, regeneration and hepatocyte transplantation.