Abstract
Fresh human hepatocytes are still considered as the “gold standard” to screen in vitro for cytochrome P450 (P450) induction. However, sparse availability of good quality human liver tissue for research purposes and the demand for standardized cell populations, together with the need for proper storage of the cells not immediately required, have resulted in the development of cryopreservation techniques that provide adequate viability and plateability of hepatocytes after thawing. This study aimed at validating cryopreserved human hepatocytes as a model to investigate P450 induction. Cryopreserved cells from four different donors were plated and cultured for 48 h, followed by incubation in the presence of typical P450 inducers. During the experiments, quality of the cultured cells was monitored both physiologically and morphologically. Concomitantly, the activity of CYP1A2, 2B6, 2C9, 2E1, and 3A4 was measured together with their mRNA and protein expression. Determination of CYP1A2, 2B6, 2C9, 2E1, and 3A4 activity in control versus prototypical inducer-treated hepatocytes revealed a maximal significant mean 11.6-, 2.8-, 1.9-, 1.5-, and 9.0-fold induction over their basal expression, respectively. Protein expression analysis of these P450s confirmed these results. Moreover, a mean 44.9-, 3.5-, 3.2-, and 13.8-fold induction of CYP1A2, 2B6, 2C9, and 3A4 mRNA was observed. Our data demonstrate that cryopreserved human hepatocytes are a valuable tool to study the induction of CYP1A2, 2B6, 2C9, 2E1, and 3A4.
Footnotes
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.104.003046.
-
ABBREVIATIONS: P450, cytochrome P450; NCE, new chemical entity; DMSO, dimethyl sulfoxide; RT, room temperature; BSA, bovine serum albumin; HRP, horseradish peroxidase; HBSS, Hanks' balanced salt solution; PhRMA, Pharmaceutical Research & Manufacturers of America; QRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; TBST, Tris-buffered saline/Tween 20.
- Received November 16, 2004.
- Accepted March 29, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|