Global gene expression changes including drug metabolism and disposition induced by three-dimensional culture of HepG2 cells-Involvement of microtubules.

Constitutive upregulation and a higher degree of induction of drug metabolism and disposition-related genes were found in a three-dimensional HepG2 culture. The upregulated genes are believed to be regulated by different regulatory factors. Global gene expression analysis using the Affymetrix GeneChip indicated that altered expression of microtubule-related genes may change the expressed levels of drug metabolizing and disposition genes. Stabilization of microtubule molecules with docetaxel, a tubulin-stabilizing agent, in the two-dimensional culture showed gene expression patterns similar to those found in the three-dimensional culture, indicating that the culture environment affects drug metabolism functions in HepG2 cells.

Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system.

Hepatitis C virus (HCV) exists in infected individuals as quasispecies, usually consisting of a dominant viral isolate and a variable mixture of related, yet genetically distinct, variants. A prior HCV infection system was developed using human hepatocellular carcinoma cells cultured in the three-dimensional radial-flow bioreactor (RFB), in which the cells retain morphological appearance and their differentiated hepatocyte functions for an extended period of time. This report studies the selection and alteration of the viral quasispecies in the RFB system inoculated with pooled serum derived from HCV carriers. Monitoring the viral RNA and core protein in the culture supernatants, together with nucleotide sequencing of hypervariable region 1 of the HCV genome, demonstrated that (1) the virus production intermittently fluctuated in the cultures, (2) the viral genetic diversity was markedly reduced 3 days post-infection (p.i.), and (3) dominant species changed on days 19-33p.i., suggesting that the virus populations can be selected according to susceptibility to the viral infection and replication. A therapeutic effect of interferon-alpha also demonstrated the inhibition of HCV expression. Thus, this HCV infection model in the RFB system should be useful for investigating the dynamic behavior of HCV quasispecies in cultured cells and evaluating anti-HCV compounds.

Gene expression property of high-density three-dimensional tissue of HepG2 cells formed in radial-flow bioreactor.

In our previous study, we examined three-dimensional culture using 5-ml radial-flow bioreactor (RFB) and showed that genes encoding cell cycle related proteins were suppressed in a stable phase. In this study, we analyzed the gene expression profiles of RFB-cultivated HepG2 cells and found that vascular endothelial growth factor (VEGF) production was strongly induced in the stable phase compared with the growth phase or static two-dimensional culture. When human umbilical vein endothelial cells (HUVECs) were grown under the conditioned medium of the stable phase, it was found that the formation of new blood vessels was induced in the angiogenesis model. DNA microarray analysis showed that the expression levels of both genes related to cell cycle arrest and which are known as tumor markers have increased in the stable phase. This result suggests that HepG2 cells in the stable phase maintain an active tumor phenotype. In addition, the expression of genes induced in the hypoxic condition was also induced in the stable phase. When the culture was carried out under a higher dissolved oxygen (DO) concentration, VEGF production did not decrease significantly and the new blood-vessel-forming ability of the conditioned medium was not suppressed. This suggests that the induction of VEGF production in a stable phase is not affected by DO during the tested level. These results suggest that the RFB cell culture system may be used to assess tumor progression mechanism under three-dimensional condition in vitro.

Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver.

A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10(8) cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5'-bromo-2'-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.

Inhibition of chromosome separation in fertilized starfish eggs by kalihinol F, a topoisomerase I inhibitor obtained from a marine sponge.

Kalihinol F, a naturally occurring diterpene from a marine sponge, Acanthella sp., inhibited chromosome separation in fertilized starfish (Asterina pectinifera) eggs but allows the first cleavage to occur, thereby forming unseparated metaphase chromosomes which were elongated between the two daughter cells. The chromosomes were eventually torn off in the embryonic cells. Most of the cells gradually lost the chromosomes during the cell cycle progression. The embryonic development halted at the morula stage just before the onset of blastulation. The mitotic failure occurred when kalihinol F was applied to a fertilized egg during the second meiotic process, but not after the completion of the second meiotic division. Kalihinol F inhibited topoisomerase I activity in vitro, but had no effects on activities of DNA polymerases alpha, beta, and gamma, and of topoisomerase II. These results suggest that the topoisomerase I plays an essential role in meiosis II in this species.