Assessment of In Vitro Applicability of Reversibly Immortalized NKNT-3 Cells and Clonal Derivatives

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Abstract:

In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4–20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.

Keywords: Hepatocyte; Immortalization; Liver; SV40 large T antigen; Telomerase

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000006783981873

Affiliations: 1: Surgical Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands, AMC Liver Center, Academic Medical Center, University of Amsterdam, The Netherlands 2: AMC Liver Center, Academic Medical Center, University of Amsterdam, The Netherlands 3: INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, Rennes, France 4: Surgical Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands

Publication date: May 1, 2006

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.

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