Immortalized hepatocytes

Immortalized Hepatocytes Yield Liver Progenitor Cells Capable to Restore the Damaged Liver... 

Immortalized hepatocytes A major drawback of the use of primary hepatocytes is that hepatocytes can not be expanded in culture. To overcome this problem, researchers have embarked on the immortalization of primary hepatocyte cultures (e.g. Bayad et al. 1991 Li et al. 2005). Currently, immortalized human hepatocyte cell lines are available commercially and may represent convenient screening experimental systems for enzyme induction studies. Unfortunately, at the time of this writing, there are no peer-reviewed publications on the application of human immortalized hepatocytes in induction studies. It is important to ensure that the induced isoforms in the cell lines are the mature P450 isoforms rather than the embryonic forms. For instance, the use of HepG2 cells may not be appropriate as the embryonic P450 isoforms CYP1A1... 

Schumacher I K, Okamoto T, Kim B H, et al. (1996). Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy. Hepatol. 24 337-743. 

Nakamura J, Okamoto T, Schumacher IK, et al. (1997). Treatment of surgically induced acute liver failure by transplantation of conditionally immortalized hepatocytes. Transplant. 63 1541-1547. 

CYP, cytochrome P450 PAMPA, parallel artificial membrane permeability assay Caco-2, human colonic adenocarcinoma cell MDCK, Madin Darby canine kidney cell rCYP, recombinant cytochrome P450 HLM, human liver microsomes PXR-TA, pregnane X receptor transactivation Fa2N-4, Fa2N-4 cryopreserved immortalized hepatocytes P-gp, P-glycoprotein. 

Amicone L, Spagnoli FM, Spath G, Giordano S, Tommasini C, Bernardini S et al (1997). Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes. EMBOJIQ 495-503. 

Mikula M, Fuchs E, Huber H, Beug H, Schulte-Hermann R, Mikulits W (2004). Immortalized pl9ARF null hepatocytes restore liver injury and generate hepatic progenitors after transplantation. Hepatology39 628-634. 

Hariparsad, N., Carr, B.A., Evers, R. and Chu, X. (2008) Comparison of immortalized Ea2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction. Drug Metabolism and Disposition The Biological Fate of Chemicals, 36, 1046-1055. 

Ripp, S.L., Mills, J.B., Eahmi, O.A., Trevena, K.A., Liras, J.L., Maurer, T.S. and de Morals, S.M. (2006) Use of immortalized human hepatocytes to predict the magnitude of clinical drug-drug interactions caused hy CYP3A4 induction. Drug Metabolism and Disposition The Biological Fate of Chemicals, 34, 1742—1748. 

To be useful to both, clinicians and the pharmaceutical industry, a bioartificial liver will need to maintain a large number of hepatocytes at high cell densities and in a fully differentiated state for prolonged periods of time. Development of such a system has been impeded by three principal problems a) a requirement for large numbers of cells (>25 10 ) b) loss of liver-specific functions in cultured cells (primary and immortalized) c) nutrient and waste product gradients in high density cultures leading to lowered cell viability and impaired function. 

Immortalization of human hepatocytes would help to overcome the hmited availability of human cells, thereby avoiding the use of mahgnant-derived cell hnes however, care will still be required with these cells. A better approach would be to develop methods for the culture of primary human hepatocytes using hormonally defined media containing growth factors in which cells are stimulated to undergo division. 

The in vitro assays described below can be run in a cell-free or cell-based manner. It should be obvious that for cell-based assays, the result will be dependent on the type of cell used, the media to which the cell is exposed and the developmental stage of the cell. Although all cells from a given individual share the same DNA, genes and pathways are turned on in a cell type-specific manner. For instance, xenobiotic metabolizing enzymes are expressed at high levels in hepatocytes, but not in most epithelial cells. Cell types are characterized as primary (taken directly from an animal and used immediately) or immortalized cell lines, which are typically derived from... 

Li J, Li LJ, Cao HC et al. (2005) Establishment of highly differentiated immortalized human hepatocyte fine with simian virus 40 large tumor antigen for liver based cell therapy. ASAIO J 51(3) 262-268... 

Some cells, such as hepatocytes, must be used in primary culture since they will not divide in culture and are relatively short-lived, while other cell lines are capable of division and can, in suitable media, be maintained indefinitely. In other cases, cells have been immortalized by fusion with tumor cells and thereafter retain the ability to divide in culture while, at the same time, maintaining many of the properties of the original nontumor cells. All of the various approaches to the use of cultured cells in biochemical and molecular toxicology are summarized in Chapter 8. The relatively recent union of the techniques of cell and molecular biology has been enormously productive for experimental toxicology since cells can be used for the expression of genetic constructs, reproduction of recombinant enzymes, and so on. 

Tsuruga Y, Kiyono T, Matsushita M, Takahashi T, Kasai H, Matsumoto S, Todo S (2008) Establishment of immortalized human hepatocytes by introduction of HPV16 E6/ E7 and hTERT as cell sources for liver cell-based therapy. Cell Transplant 17(9) 1083-1094... 

Zern MA (2003) Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 124(2) 432-444 58. Kobayashi N, Noguchi H, Westerman KA, Watanabe T, Matsumura T, Totsugawa T, Fujiwara T, Leboulch P, Tanaka N (2001) Cre/loxP-based reversible immortalization of... 

Youdim, K. A., Tyman, C. A., Jones, B. C., and Hyland, R. (2007) Induction of cytochrome P450 assessment in an immortalized human hepatocyte cell line (Fa2N4) using a novel higher throughput cocktail assay. Drug Metab. Dispos. 35, 275-282. 

Naoya Kobayashi, Toshiyoshi Fujiwara, Karen A. Westerman, Yusuke Inoue, Masakiyo Sakaguchi, Hirofumi Noguchi, Masahiro Miyazaki, Jin Cai, Noriaki Tanaka, Ira J. Fox, Philippe Leboulch, Prevention of Acute Liver Failure in Rats with Reversibly Immortalized Human Hepatocytes, Science, 287 (2000), 1258-1262. 

Kobayashi N, Fujiwara T, Westerman K A, et al. (2000). Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes. Science. 287 1258-1262. 

Okitsu T, Kobayashi N, Jun H S, et al. (2004). Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs. Diabetes. 53 105-112. 

Kobayashi N, Miyazaki M, Fukaya K, et al. (2000). Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure. Transplant. 69 202-207. 

Blumrich M, Zeyen-Blumrich U, Pagels P, et al. (1994). Immortalization of rat hepatocytes by fusion with hepatoma cells. II. Studies on the transport and synthesis of bile acids in hepatocytoma (HPCT) cells. Eur. J. Cell Biol. 64 339-347. 

Fox I J, Chowdhury N R, Gupta S, et al. (1995). Conditional immortalization of Gunn rat hepatocytes an ex vivo model for evaluating methods for bilirubin-UDP-glucuro-nosyltransferase gene transfer. Hepatol. 21 837-846. 

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