Primary human hepatocytes

Plainly DILI is a major cause for concern toward which efforts to detect potential hepatotoxicants before they reach later stages of development should be made. During development, molecules must be tested using in vitro models before progressing to animal and human trials. There are several in vitro models available including primary human hepatocytes, inunortalized cell lines and animal models, and, the main subject of this chapter, pluripotent stem cell-derived hepatocyte-like cells (HLCs). 

Dedifferentiation begins as soon as the tissue becomes ischemic during resection and is worsened during isolation by induction of ischemia-reperfusion injury when the tissue is reperfused and use of collagenase that is typically contaminated with Upopolysaccharide producing an inflammatory stress response (Wang et al., 1998 Elaut et al., 2006). Dedifferentiation is characterized by the loss of 

Reference Stem cell type Culture format Differentiation factors % ALB -i-ve HLCs (assay) Enzyme (assay method) %hPH comparator Other comparators 

Chen et al. hESC (H9), hiPSC Monolayer AA,rrS,HGF,Wnt3A, — CYP3A4 (bioluminescence) 100 hiPSC 

APPLICATION OF PLURIPOTENT STEM CELLS IN DRUG-INDUCED LIVER INJURY SAFETY ASSESSMENT 

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Misra A, Hong JY, Kim S (2007) Multiplex genotyping of cytochrome p450 single-nucleotide polymorphisms by use of MALDI-TOF mass spectrometry. Clin Chem 53 933-939 Moon YJ, Zhang S, Brazeau DA, Morris ME (2007) Effects of the flavonoid biochanin A on gene expression in primary human hepatocytes and human intestinal cells. Mol Nutr Food Res 51 317-323... 

Metabolic competence of HepG2 human hepatoblastoma cells depends on the source and culture conditions. They have both Phase I and II metabolizing enzymes. Cytochrome P450 enzymes are found in much lower levels in HepG2 cells than in primary human hepatocytes but many of these enzymes are inducible, including CYPlAl, 1A2, 2B6, 2E1 and 3A4. The latter metabolizes approximately 50% of drugs currently on the market [32]. 

Westerink, W.M.A. and Schoonen, W.G.E.J. (2007) Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology In Vitro, 21, 1581-1591. 

Table 15.1 The high content cellular imaging data from the primary human hepatocyte cultures, upon overnight treatment by a panel of 30 selected drugs. 

Immortalization of human hepatocytes would help to overcome the hmited availability of human cells, thereby avoiding the use of mahgnant-derived cell hnes however, care will still be required with these cells. A better approach would be to develop methods for the culture of primary human hepatocytes using hormonally defined media containing growth factors in which cells are stimulated to undergo division. 

The effect of botanical products on the expression of drug-metabolizing enzymes or transport proteins can be examined in cell culture with established cell lines or primary human hepatocytes. The cells are incubated under... 

Faucette SR, Wang H, Hamilton GA, et al. Regulation of CYP2B6 in primary human hepatocytes by prototypical inducers. Drug Metab Dispos 2004 32(3) 348-358. 

Primary human hepatocytes in particular have not yet proven to be compatible with cryopreservation (Utesch et al., 1992), therefore studies must be performed when the tissue becomes available. Characterization of the enzyme composition of the tissue derived from an individual must be conducted in parallel with characterization of the unknown xenobiotic. This can lead to the devotion of considerable experimental effort to studies which, in the end, do not meet quality control criteria. Despite these limitations, human hepatocytes are uniquely suited for studies of cytochrome P450 regulation and also provide the only current system which maintains a balanced and physiological ratio of cofactors and individual Phase I and Phase II enzymes. 

Drocourt L, Ourlin JC, Pascussi JM, Maurel P, Vilarem MJ. Expression of CYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes. J Biol Chem 2002 277 25125-25132. 

Chang TK, Yu L, Maurel P, et al. Enhance cyclosphosphamide and ifosfamide activation in primary human hepatocyte cultures response of cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res 1997 57 1946-1954. 

Li AP, Hartman NR, Lu C, et al. Effects of cytochrome P450 inducers on 17alpha-ethinyloestradiol (EE2) conjugation by primary human hepatocytes. Br J Clin Pharmacol 1999 48 733-742. 

Li AP, Reith MK, Rasmussen A, et al. Primary human hepatocytes as a tool for the evaluation of structure-activity relationship in cytochrome P450 induction potential of xenobiotics evaluation of rifampin, rifapentine and rifabutin. Chem Biol Interact 1997 107 17-30. 

Thogersen IB, Enghild JJ. Biosynthesis of bikunin proteins in human carcinoma cell line HepG2 and in primary human hepatocytes. J Biol Chem 1995 270 18700-18709. 

Typical results for the testing of a test article for CYP1A and CYP3A induction using primary human hepatocytes are shown in Table 5. 

Harris AJ, Dial SL, Casciano DA (2004) Comparison of basal gene expression profiles and effects of hepatocarcinogens on gene expression in cultured primary human hepatocytes and HepG2 cells. Mutat Res 549 79-99... 

Li AP (1997) Evaluation of drug-drug interactions in primary human hepatocytes. In Li AP (ed) Drug-drug Interactions Scientific and Regulatory Perspectives. Advances in Pharmacology 43 103-130... 

Li AP, Maurel JP, Lechon-Gomez M et al. (1997) Preclinical evaluation of drug-drug interactions present status of the application of primary human hepatocytes in the evaluation of cytochrome P450 induction. Chemico-Biological Interactions 107 5-16... 

Li AP, Reith MK, Rasmussen A et al. (1997) In vitro evaluation of drug-drug interaction potential A comparison of rifampin, rifapentine, and rifabutin in cytochrome P450 3A induction potential in primary human hepatocytes. Chemico-Biological Interactions 107 17-30... 

Soars MG, Petullo DM, Eckstein JA et al. (2004) An assessment of udp-glucuronosyltransferase induction using primary human hepatocytes. Drug Metab Dispos 32 140-148... 

Olsavsky KM. 2007. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues. Toxicol. Appl. Pharmacol. 222,42-56 Epub Apr. 21 2007. 

Wifkening S. 2003. Comparison of primary human hepatocytes and hepatoma cell fines HepG2 with regard to their biotransfomation properties. Drug Metabol. Dispos. 31, 1035-1042. 

Tang, L. Komoroski, B. Tiegler, K. Tibbits, J. Nolan, T. Suizu, R. Neft, R. Kier, L. Strom, S. Li, A.P. A comparison of array hybridization, mRNA differential display and real-time PCR in the evaluation of the effects of Troglita-zone on gene expression in primary human hepatocytes. Toxicologist 2003, 72 (SI), 149. 

Wilkening, S. Bader, A. Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line FlepG2. J. 

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