Well maintained expression of CYP genes in sandwich-culturing hepatocytes: quantitative analysis using real-time PCR method.
Sandwich-culturing is an excellent hepatocyte culturing method in drug metabolism studies, however, its advantages for gene expression of cytochrome P450 (CYP) have not been evaluated so far. The present study was undertaken to determine the utilities of sandwich-culturing hepatocytes for evaluation of CYP genes expression. Hepatocytes from male rats were cultured for 5 days between two layers of type-I collagen gel (sandwich-culturing) or over type-I collagen gel (single gel culturing). To determine the expression of CYP genes rapidly and accurately, the time course study using real-time RT-PCR quantification was conducted in the present study, and CYP2B1, CYP2B2, CYP3A2, CYP3A9 and CYP3A23 genes were measured. Albumin secretion was also measured by ELISA to evaluate cell viability. Higher expression and excellent maintenance of all CYP genes were confirmed in sandwich-culturing hepatocytes than those in single gel culturing. Particularly, significant difference in the amounts of CYP genes expression was observed between both methods after 3 days culturing. Albumin secretion was also higher in sandwich-culturing after 3 days culturing, suggesting that the cell viability of hepatocytes was maintained. These results indicate that sandwich-culturing method is much more advantageous than the ordinate method in maintaining the CYP gene expression and cell viability.
Toritsuka
N
N
Drug Safety and Disposition Research Laboratories, Eisai Co., Ltd., 1 Takehaya-machi, Kawashima-cho, Gifu, 501-6195, Japan. n-toritsuka@hhc.eisai.co.jp
Nonogaki
Y
Y
Hori
Y
Y
Sawada
S
S
Shoun
H
H
Motooka
S
S
D000818:Animals; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D022781:Hepatocytes / Q000378:metabolism*; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003577:Cytochrome P-450 Enzyme System