A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers.

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

Increase of ß2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane.

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. ß-actin, ß-catenin, and ß1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but ß2-integrin expression markedly increased.

[Selective elimination of the transformed hepatic stem cells using hybrid liposomes].

To realize regenerative medicine, it is very important to eliminate the transformed stem cells selectively included in iPS cells, ES cells and adult stem cells derived from organs, because the transformed stem cells have a risk of tumorigenesis after the cell transplantation. Ueoka et al., have developed hybrid liposomes (HL) which selectively accumulated to membranes of tumor cells and have high inhibitory effects on the growth of tumor cells along with the induction of apoptosis. Therefore, we have investigated the application of HL23 (DMPC/10 mol%C(12)(EO)(23)) to the selective elimination of transformed stem cells using hepatoblast, which we could induce from human fetal hepatocytes by the treatment of 1 mM sodium butyrate for 8 days. During the induction process, the transformed cells appeared and produced abnormal prothrombin (PIVKA-II), which is a clinical marker for hepatoma, and also formed colonies in soft agar plate, which is a criteria for neoplastic cell transformation. On the other hand, by the treatment with 0.33 mM HL23 for 96 h during the induction process, PIVKA-II production rate of the cells and colonies formed in the soft agar plate also remarkably decreased less than those of the normal cells. Furthermore, the population of hepatoblasts in the remaining cells increased about four times. These results suggest that the transformed hepatic stem cells could be selectively eliminated by the treatment of HL23, and HL treatment of the stem cells would be a useful culture method for quality control of the stem cells to reduce a risk of tumorigenesis after the cell transplantation.

Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells.

Human hepatic cancer cells, HepG2, formed spheroids on a poly-L-glutamic acid-coated dish. Doxorubicin (DOX) efflux activity of the cells in spheroid culture was higher than that in monolayer culture due to the higher expression of MDR1 protein of the cells in spheroids compared with those in monolayer. The amount of MDR1 per cell in spheroids was similar to that of hepatic tumor tissue in vivo. Consequently, it was suggested that the drug efflux activity of cells in spheroid culture reflected the activity of hepatic cancer cells. Furthermore, the IC(50) of DOX and epirubicin (EPI) in HepG2 cells, both of which are known to be exported by MDR1, were higher in spheroid compared with monolayer cells, while IC(50) of 5-fluorouracil (5-FU), which is not exported by MDR1 protein, was almost the same in both types of culture. The higher IC(50) of DOX and EPI in HepG2 cells in spheroid culture was associated with a higher efflux activity of the drugs in the spheroid-cultured cells, which appeared to reflect the IC(50) of DOX and EPI in cancer cells in vivo. Therefore, a spheroid culture of hepatic cancer cells seems to provide a promising cell-based in vitro assay system for examining the proper IC(50) values of anticancer agents that would reflect the drug resistance of cancer cells in vivo. In addition, the system would be useful in screening for inhibitors of MDR1 activity, which will help to overcome the multidrug resistance of cancer cells.