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Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

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Abstract

The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication, morphology, and function were lost if the cells were cultured without STO feeder cells. A method for the feeder-independent continuous culture of PICM-19 cells (FI-PICM-19) is presented. PICM-19 cells were maintained and grown without feeder cells on collagen I-coated tissue culture plastic for 26 passages (P26) with initial split ratios of 1:3 that diminished to split ratios of less than 1:2 after passage 16. Once plated, the FI-PICM-19 cells were overlaid with a 1:12 to 1:50 dilution of Matrigel or related extracellular matrix product. Growth of the cells was stimulated by daily refeedings with STO feeder-cell conditioned medium. The FI-PICM-19 cells grew to an approximate confluence of 50% prior to each passage at 2-wk intervals. Growth curve analysis showed their average cell number doubling time to be ~96 h. Morphologically, the feeder-independent cells closely resembled PICM-19 cells grown on feeder cells, and biliary canalicui were present at cell-to-cell junctions. However, no spontaneous multicellular ductules formed in the monolayers of FI-PICM-19 cells. Ultrastructural subcellular features of the FI-PICM-19 cells were similar to those of PICM-19 cells cultured on feeder cells. The FI-PICM-19 cells produced a spectrum of serum proteins and expressed many liver/hepatocyte-specific genes. Importantly, cytochrome P450 (EROD) activity, ammonia clearance, and urea production were maintained by the feeder-independent cells. This simple method for the propagation of the PICM-19 cell line without feeder cells should simplify the generation and selection of functional mutants within the population and enhances the cell line’s potential for use in toxicological/pharmacological screening assays and for use in an artificial liver device.

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Acknowledgments

We thank Amy Shannon for assistance with enzyme assays, Lori Schreier for assistance with real-time RT-PCR, and Paul Graninger for his diligence with two-dimensional gel protein analysis, cell metabolic assays, and cell enzyme activity assays. Mention of trade names or commercial products in this publication is solely for the purposes of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

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Authors and Affiliations

  1. U. S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Bldg. 200, Rm. 13, BARC-East, Beltsville, MD, 20705, USA

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

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  1. Neil C. Talbot
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  2. Le Ann Blomberg
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  3. Wesley M. Garrett
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  4. Thomas J. Caperna
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Correspondence to Neil C. Talbot.

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Editor: J. Denry Sato

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Talbot, N.C., Blomberg, L.A., Garrett, W.M. et al. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line. In Vitro Cell.Dev.Biol.-Animal 46, 746–757 (2010). https://doi.org/10.1007/s11626-010-9336-9

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  • DOI: https://doi.org/10.1007/s11626-010-9336-9

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