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.

We previously reported that using thermo-responsive culture surfaces, a layered co-culture was achieved by placing an endothelial cell sheet onto a layer of human hepatoma cell line HepG2 in order to develop a culture model that mimics hepatic lobules. In the layered co-culture cells, the expression levels of liver-specific genes gradually increased. A cross-sectional view of the layered co-culture cells showed that the thickness of the layer slowly increased after layering, as did extracellular matrix (ECM) deposition around HepG2 cells. In this study, we report that the molecular compositions of ECM and cell-adhesion molecules changed in the layered co-culture cells. Gene expression of integrin alpha4 and decorin gradually increased after layering, and the time-course pattern of these genes was correlated with that of liver-specific genes. Moreover, the layered co-culture system has the ability to assemble a branching network of fibronectin fibrils. These results suggest that a vastly different extracellular environment in layered co-culture cells may induce an increase in liver-specific functions.

Primary culture of human hepatocytes is a useful in vitro system to study the potential of drugs to induce cytochrome P450 (CYP) 1,2) and to analyze the activity and regulation of drug uptake and efflux transporters. [3][4][5] Indeed, human hepatocytes exhibit the entire repertoire of hepatic drug-metabolizing enzymes, drug transporters and the genes for liver-specific functions. 6,7) However, the expression of many proteins has been shown to decrease rapidly in cultured primary hepatocytes, with drug metabolizing enzymes being particularly sensitive.8) Liver slices retain metabolic function longer than primary hepatocytes, 9) however drug metabolism decreases rapidly upon incubation, and the erratic supply of human liver tissue is a major problem.The human hepatocyte cell line HepG2 is also a useful in vitro model for investigation of the metabolism and toxicity of drugs, as these cells retain many of the specialized functions which are characteristic of normal human hepatocytes and are easy to manipulate. 10,11) However, the gene expression levels and activity of drug-metabolizing enzymes in this cell line are lower than in primary hepatocytes, [12][13][14] and induction of the CYP genes by inducers is not observed in HepG2 and other cell lines. 15,16) Thus, a more functional culture system is required to obtain the usually high levels of activity and regulation ability of drug metabolizing systems.Previously we reported the development of a culture surface produced using a thermo-responsive polymer, poly(Nisopropylacrylamide) (PIPAAm). 17,18) Using this thermo-responsive surface, we assembled a layered co-culture system consisting of hepatic cells and sheets of endothelial cells, which allows for continuous expression of the differentiated functions of hepatocytes. In addition, expression of albumin and apolipoprotein A-1 increases 19) and numerous hepatocyte and endothelial cell functions increase in intensity upon a layered co-culture system. 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. The gene expression of CYP2C and CYP3A and activity of CYP3A4 were induced by phenobarbital in a concentration-dependent manner. Furthermore, the induction of hepatic drug transporters was detected in the layered co-cultured HepG2 cells. These results indicate that the functions of CYP and drug transporters are retained in the layered co-cultured cells, making it a useful model for drug metabolism analysis. 22) were cultured in Dulbecco's modified...