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

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Abstract

The establishment and initial characterization of bovine fetal liver cell lines are described. Bovine fetal hepatocytes were cultured from the liver of a 34-d bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO (SIMS mouse strain, thioguanine- and ouabain-resistant) feeder layers and were cultured in a medium supplemented with 10% fetal bovine serum. After 2–3 wk, 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 further 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 transforming growth factor (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. 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 cyclic adenosine monophosphate (cAMP)-stimulating chemicals 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.

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Acknowledgments

We thank Amy Shannon for her technical expertise in the urea assay and are grateful for the editorial and scientific comments of Dr. Le Ann Blomberg. Care and treatment of cows in this study were approved by the Institutional Animal Care and Use Committee of the U.S. Department of Agriculture.

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  1. Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, NEA, ARS, USDA, Bldg. 200, Rm. 13, BARC-East, Beltsville, MD, 20705, USA

    Neil C. Talbot, Wesley M. Garrett & Thomas J. Caperna

  2. Department of Animal Science, University of Connecticut, Storrs, CT, 06269, USA

    Ling Wang & Young Tang

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  1. Neil C. Talbot
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  2. Ling Wang
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Correspondence to Neil C. Talbot.

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Talbot, N.C., Wang, L., Garrett, W.M. et al. Establishment and characterization of feeder cell-dependent bovine fetal liver cell lines. In Vitro Cell.Dev.Biol.-Animal 52, 314–326 (2016). https://doi.org/10.1007/s11626-015-9982-z

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