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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #322202

Title: Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

Author
item Talbot, Neil
item WANG, LING - University Of Connecticut
item Garrett, Wesley
item CAPERNA, THOMAS - Retired ARS Employee
item TANG, YOUNG - University Of Connecticut

Submitted to: In Vitro Cellular and Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2015
Publication Date: 12/10/2015
Citation: Talbot, N.C., Wang, L., Garrett, W.M., Caperna, T.J., Tang, Y. 2015. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines. In Vitro Cellular and Developmental Biology. 52(3):314-26.

Interpretive Summary: The establishment and initial characterization of bovine fetal liver (BFL) cell lines is described. The bovine liver cells multiplied in the culture dish and could be transferred to new culture dishes for further growth. The BFL cells could be grown for about one year before they suffered from aging and stop dividing. The BFL cell cultures maintained many of the key liver functions that they have if they were still in the body of the cow. The culture system provides a good in vitro model of fetal bovine liver cells and is the first culture of normal bovine liver cells that can be grown for a long time. The BFL in vitro model will enable experiments to be done to define better bovine liver function, bovine liver responses to diseases and parasites, and the modeling of how genetic changes affect bovine liver functions.

Technical Abstract: The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and were culture in medium supplemented with 10% fetal bovine serum. After 2-3 wks, primary colonies of hepatocytes were observed by phase-contrast microscopic observation. Individual hepatocyte colonies were colony-cloned into independent bovine fetal liver (BFL) cell lines. Two cell lines, BFL-6 and BFL-9, grew the best of several isolates, and they were characterized for growth potential and for hepatocyte morphology and function. The two cell lines were found to grow markedly better in the presence of the TGF-beta inhibitor, SB431542 (1 µM). Their continuous culture also depended on a particular medium height - for T12.5 flasks, 3 ml total medium produced optimum growth. Higher or lower amounts of medium caused less cell growth or cessation of growth and cell die off. The cell lines were propagated for over a year at split ratios of 1:2 or 1:3 at each passage until reaching senescence at approximately 30 passages. The cells were laterally polarized with well developed canalicular spaces occurring between adjacent BFL cells. Treatment of the cultures with cAMP-stimulating substances or peptides (e.g., forskolin or glucagon) caused physical expansion of the canaliculi between the cells within 15 min. The cells secreted a spectrum of serum proteins, were positive for the expression of several hepatocyte-specific genes and converted ammonia to urea, although at a relatively low rate. The culture system provides an in vitro model of fetal bovine hepatocytes and is the first demonstration of the continuous culture of normal bovine hepatocytes as cell lines.