Abstract
Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study, we investigated whether cryopreserved SHs could express cytochromes P450 (P450s), whether P450 expression was induced by appropriate inducers, and whether P450 activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16α-carbonitrile (PCN), and ethanol were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3-MC and PCN, respectively. Although Matrigel did not up-regulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs, although the activities in both were approximately 30% of that of MHs. Profiles of 14C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by high-performance liquid chromatography. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of P450s after long-term cryopreservation.
Footnotes
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This study was supported by grants from the Science and Technology Incubation Program in Advanced Region, the Japan Science and Technology Agency, the Ministry of Education, Culture, Sports, Science and Technology, Japan (14370393, 17390353) to T.M., and Ministry of Health, Labour and Welfare, Health and Labour Sciences Research Grants, Research on Advanced Medical Technology to T.M.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.008342.
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ABBREVIATIONS: P450, cytochrome P450; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; EtOH, ethanol; HPLC, high-performance liquid chromatography; KH, Krebs-Henseleit; 3-MC, 3-methylcholanthrene; MH, mature hepatocyte; PB, phenobarbital; PCN, pregnenolone-16α-carbonitrile; SH, small hepatocyte.
- Received November 16, 2005.
- Accepted July 19, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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