Up-Regulation of Drug-Metabolizing Enzyme Genes in Layered Co-Culture of a Human Liver Cell Line and Endothelial Cells

Abstract

Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation.

Induction of Drug-Metabolizing Enzymes by Phenobarbital in Layered Co-culture of a Human Liver Cell Line and Endothelial Cells

“…In our previous study, the CYP3A4 activity in the layered co-cultured cells increased 3.4-fold compared to that in the monolayer culture cells. 21) These results indicate that the increased expression of the cytochrome P450 mRNAs correlates with increased CYP enzyme activity.…”

“…15 previously reported that in the layered co-cultured cells, consisting of a BPAEC sheet overlaid onto HepG2 cells, the gene expression levels of CYP2C9, CYP3A4 and CYP3A7 were significantly increased compared with monolayer cultured HepG2 cells. 21) The results suggested that our co-culture system would be suitable for drug metabolism analysis.…”

“…13) We previously reported that in the layered co-cultured HepG2 cells, the gene expression level of the CYP3A4 was about 100 times greater than monolayer cultured HepG2 cells. 21) In the present study, we measured the CYP3A4 activity in the layered co-cultured HepG2 cells. Figure 3 shows the relative CYP3A4 activity in the layered co-culture at 21 d after overlaying the BPAEC sheet onto hepatic HepG2 cells.…”

“…The 3-dimensional manipulation of the BPAEC sheets and assembly of the layered co-cultured cells were performed as previously described. [19][20][21] Briefly, BPAECs were plated on the UpCell dishes at a cell density of 1.0ϫ10 6 cells/dish and cultured for 1 week at 37°C. After removing the culture medium, a sheet of polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, U.S.A.) was placed over the BPAEC monolayer.…”

“…20) We also reported that in a layered co-culture system with HepG2 cells and bovine pulmonary artery endothelial cells (BPAECs) forming a sheet, the gene expression levels of various CYP and other drugmetabolizing enzymes were significantly increased, compared with the monolayer cultured HepG2 cells, and were maintained at high levels for extended periods. 21) The present study analyzed the drug metabolism of this layered co-culture system. To study the induction of CYP expression by an inducer in the co-culture system, we analyzed the effect of phenobarbital treatment on CYP expression in the layered co-cultured HepG2 cells.…”