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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Chen, Liqiong (2010)
Languages: English
Types: Unknown
Subjects:
The development of systems for the long term in vitro culture of functional liver tissue is a major research goal. The central limitation of experimental systems to date has been the early de-differentiation of primary hepatocytes in cultures. Several factors including cell-cell interaction, cell-matrix interaction, soluble factors and 3D structures have been identified as the keys to overcome this limitation. The first aim of this project is to compare the established 3D model, co-culture of hepatocytes and hepatic stellate cells (HSCs) on PDLLA coated surfaces, to other best available systems using collagen and Matrigel. The hypothesis is that hepatocytes functionalities, established by cell-cell interaction, 3D structures and soluble factors, can be further enhanced by introduction of cell-matrix interaction. In order to test the hypothesis, rat hepatocytes were cultured in five different systems, including monoculture of hepatocytes on collagen gel, in collagen-Matrigel sandwich, co-culture of hepatocytes and HSCs on collagen gel, in collagen-Matrigel sandwich and on PDLLA coated surface. Hepatocyte specific function assays, namely albumin secretion, urea secretion, testosterone metabolism by HPLC and CYP activities by LC-MS-MS, were used to analyze cell functionalities. Homo-spheroids were only formed in monoculture on collagen gel, but hetero-spheroids were developed in all the co-culture systems. The results of function assays showed hepatocytes in collagen-Matrigel sandwich configuration had the best secretion of albumin and urea and best CYP activities during the culture period. These data demonstrated the hypothesis that hepatocyte functions of the established model can be further improved by introduction of cell-matrix interaction. In addition to establishment of rat hepatocyte culture systems, hetero-spheroids of primary human hepatocytes and primary human HSCs on PDLLA coated plates were developed successfully, due to the great improvements of isolation and culture of primary human HSCs. However, hepatocyte function assays have not been applied yet. Hepatic cell lines have several advantages that are not applicable to primary cultured human hepatocytes, namely unlimited lifespan and stable phenotype. The immortalized Fa2N-4 cell lines have recently been assessed as replacements of primary human hepatocytes in CYP induction studies. The second aim of this study was to simultaneously characterize CYP1A2, CYP2C9, CYP3A4 and CYP2B6 induction in Fa2N-4 cells through assessment of mRNA, protein and activity endpoints for a range of prototypical compounds (previously assessed in human hepatocytes) with known positive and negative induction potential. LC-MS-MS and RT-PCR were used for assessment of activity and mRNA endpoints respectively. As a result, it is considered that Fa2N-4 cells offer a substitute for primary human hepatocytes for CYP1A2 and CYP3A4 induction but not for CYP2B6 due to lack of cytosolic CAR expression.
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