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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF STEM CELL AND NUCLEAR CLONING TECHNOLOGIES IN UNGULATES Title: Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

Authors
item CAPERNA, THOMAS
item BLOMBERG, LE ANN
item GARRETT, WESLEY
item TALBOT, NEIL

Submitted to: In Vitro Cellular and Developmental Biology - Animals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2010
Publication Date: March 1, 2011
Citation: Caperna, T.J., Blomberg, L., Garrett, W.M., Talbot, N.C. 2011. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media. In Vitro Cellular and Developmental Biology - Animals. 47(3):218-233.

Interpretive Summary: The study presents a method for the selective in vitro culture, i.e., “in the petri dish,” of pig hepatocytes and bile duct cells, i.e., liver cells. The report characterizes the cells general health and typrical in vivo-like, i.e., “in the body-like,” appearance and functions. Also, presented are data on specific liver gene expression and liver serum-protein production that again show that the hepatocytes and bile duct cell cultures are similar to liver cells found in a pig’s own liver. For agricultural purposes, because the liver is so important to the growth and maintenance of the pig, this in vitro model could be useful for testing man-made genetic changes to the liver function of pigs prior to the actual genetic engineering of the pig, and it could be useful for the in vitro testing of pig liver physiology. The cell culture system could also be useful for biomedical purposes. For example, the pig liver cell cultures could be used as the biological component, i.e., live cell component, of an artificial liver device for the treatment of people suffering from acute liver failure, and for pharmacological and toxicological assays of liver function and liver damage.

Technical Abstract: A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 µM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several weeks. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder-cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiiocyte monolayers. Serum protein expression, analyzed by 2D-gel electrophoresis of conditioned medium, was remarkably similar between the two cell culture conditions. However, quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte form and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.

Last Modified: 9/10/2014
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