Skip to main content
SpringerLink
Log in

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

  • Cellular Models
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4–8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7–10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Alpini, G.; Phillips, J. O.; LaRusso, N. F. The biology of biliary epithelia. In: Arias, I. M.; Boyer, J. L.; Fasto, N., et al., ed. The liver: biology, and pathobiology. 3rd edition. New York: Raven Press; 1994:623–653.

    Google Scholar 

  • Bader, A; Christains, U.; Boker, K., et al. In vitro imitation of the in vivo three dimensional microenvironment enables primary hepatocytes to maintain stable metabolic functions. Cell Transplant. 1:162; 1992.

    Google Scholar 

  • Baumann, H.; Wong, C. G. Hepatocyte-stimulating factor III shares structural and functional identity with leukemia-inhibitory factor. J. Immunol. 143:1163–1167; 1989.

    PubMed  CAS  Google Scholar 

  • Caperna, T. J.; Failla, M. L.; Kornegay, E. T., et al. Isolation and culture of parenchymal and nonparenchymal cells from neonatal swine liver. J. Anim. Sci. 61:1576–1586; 1985.

    PubMed  CAS  Google Scholar 

  • Daoust, R.; Cantero, A. The numerical proportions of cell types in rat liver during carcinogenesis by 4-dimethylaminoazobenzene (DAB). Cancer Res. 19:757–762; 1959.

    PubMed  CAS  Google Scholar 

  • Dawes, C. J. Biological techniques in electron microscopy. Burlington, Ladd Research Industries, Inc.; 1971:39.

    Google Scholar 

  • Demetris, A. J.; Markus, B. H.; Saidman, S., et al. Isolation and primary cultures of human intrahepatic bile ductular epithelium. In Vitro Cell. Dev. Biol. 24:464–470; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Dunn, J. C. Y.; Yarmush, M. L.; Koebe, H. G., et al. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J. 3:174–177; 1989.

    PubMed  CAS  Google Scholar 

  • Goldstein, J. L.; Ho, Y. K.; Basu, S. K., et al. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc. Natl. Acad. Sci. USA 76:335–337; 1979.

    Google Scholar 

  • Grant, A. G.; Billing, B. H. The isolation and characterization of a bile ductule cell population from normal and bile-duct ligated rat livers. Br. J. Exp. Pathol. 58:301–310; 1977.

    PubMed  CAS  Google Scholar 

  • Grubman, S. A.; Perrone, R. D.; Lee, D. W., et al. Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines. Am. J. Physiol. 266:G1060-G1070; 1994.

    PubMed  CAS  Google Scholar 

  • Ishii, M.; Vroman, B.; LaRusso, N. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97:1236–1247; 1989.

    PubMed  CAS  Google Scholar 

  • Jacobs, W. L. W. A colorimetric assay for gamma-glutamyltranspeptidase. Clin. Chim. Acta 31:175–179; 1971.

    Article  PubMed  CAS  Google Scholar 

  • Johnston, D. E.; Marcadis, D.; Corcoran, G. B., et al. Purification of primary human hepatocytes using ricin A chain. In Vitro Cell. Dev. Biol. 32A:388–390; 1996.

    Google Scholar 

  • Joplin, R.; Stain, A. J.; Neuberger, J. M. Immuno-isolation and culture of biliary epithelial cells from normal human liver. In Vitro Cell. Dev. Biol. 25:1189–1192; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Joplin, R.; Hishida, T.; Tsubouchi, H., et al. Human intrahepatic biliary epithelial cells proliferate in vitro in response to human hepatocyte growth factor. J. Clin. Invest. 90:1284–1289; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Linsenmayer, T. F. Collagen. In: Hay, E. D., ed. Cell biology of the extracellular matrix. 2nd ed. New York: Plenum Press; 1991:7–44.

    Google Scholar 

  • Mathis, G. A.; Walls, S. A.; Sirica, A. E. Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured “on top” and “inside” different extracellular matrix substitutes. Cancer Res. 48:6145–6153; 1988.

    PubMed  CAS  Google Scholar 

  • Mathis, G. A.; Sirica, A. E. Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells. In Vitro Cell. Dev. Biol. 26:113–118; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Meister, A.; Tate, S. S.; Leonard, R. L. Membrane-bound-gamma-glutamyl transpeptidase. In: Martinosi, A., ed. The enzymes of biological membranes. New York: Plenum Press; 1976:315–347.

    Google Scholar 

  • Montesano, R.; Matsumoto, K.; Nakamura, T., et al. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67:901–908; 1991a.

    Article  PubMed  CAS  Google Scholar 

  • Montesano, R.; Schaller, G.; Orci, L. Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell 66:697–711; 1991b.

    Article  PubMed  CAS  Google Scholar 

  • Nerurkar, L. S.; Marino, P. A.; Adams, D. O. Quantification of selected intracellular and secreted hydrolases of macrophages. In: Herskowitz, H. B.; Holden, H. T.; Bellanti, J. A.; Ghaffar, A., ed. Manual of macrophage methodology. New York: Marcel Dekker, Inc.; 1981:229–247.

    Google Scholar 

  • Okamoto, H.; Ishii, M.; Mano, Y., et al. Confluent monolayers of bile duct epithelial cells with tight junctions. Hepatology 22:153–159; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Omura, T.; Sato, R. The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem. 239:2370–2378; 1964.

    PubMed  CAS  Google Scholar 

  • Paradis, K.; Sharp, H. L. In vitro duct-like structure formation after isolation of the bile ductular cells from a murine model. J. Lab. Clin. Med. 113:689–694; 1989.

    PubMed  CAS  Google Scholar 

  • Perrone, R. D.; Grubman, S. A.; Rogers, L. C., et al. Continuous epithelial cell lines from ADPKD liver cysts exhibit characteristics of intrahepatic biliary epithelium. Am. J. Physiol. 269:G335-G345; 1995.

    PubMed  CAS  Google Scholar 

  • Robertson, E. J. Embryo-derived stem cell lines. In: Robertson, E. J., ed. Teratocarcinomas and embryonic stem cells: a practical approach. Oxford, England: IRL Press; 1987:71–112.

    Google Scholar 

  • Rathjen, P. D.; Toth, S.; Willis, A., et al. Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell 62:1105–1114; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Rhim, J. A.; Sandgren, E. P.; Degen, J. L., et al. Replacement of diseased mouse liver by hepatic cell transplantation. Science 263:1149–1152; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Rohme, D. Evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo. Proc. Natl. Acad. Sci. USA 78:5009–5013; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Rutenberg, A. M.; Kim, H.; Fischbein, J. W., et al. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J. Histochem. Cytochem. 17:517–526; 1969.

    Google Scholar 

  • Schmitt, R. M.; Bruyns, E.; Snodgrass, H. R. Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev. 5:728–740; 1991.

    PubMed  CAS  Google Scholar 

  • Singh, A.; Shahidi, E. Ultrastructure of the piglet liver. In: Tumbleson, M. E., ed. Swine in biomedical research. Vol. 1. New York: Plenum Press; 1987:77–87.

    Google Scholar 

  • Sirica, A. E.; Cihla, H. P. Isolation and partial characterization of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats. Cancer Res. 44:3454–3466; 1984.

    PubMed  CAS  Google Scholar 

  • Sirica, A. E.; Sattler, C. A.; Cihla, H. P. Characterization of a primary bile ductular cell culture from the livers of rats during extrahepatic cholestasis. Am. J. Pathol. 120:67–78; 1985.

    PubMed  CAS  Google Scholar 

  • Sirica, A. E. Biology of biliary epithelial cells. Progress in Liver Diseases 10:63–87; 1992.

    PubMed  CAS  Google Scholar 

  • Sirica, A. E.; Gainey, T. W. A new rat bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro. Hepatology 26:537–549; 1997.

    PubMed  CAS  Google Scholar 

  • Stoker, M.; Gherardi, E.; Perryman, M., et al. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 327:239–242; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Talbot, N. C.; Rexroad, C. E., Jr.; Pursel, V., et al. A continuous culture of pluripotent fetal hepatocytes derived from the 8-d epiblast of the pig. In Vitro Cell. Dev. Biol. 30A:843–850; 1994a.

    CAS  Google Scholar 

  • Talbot, N. C.; Rexroad, C. E., Jr.; Pursel, V., et al. Colony-cloning and secondary culture of fetal porcine hepatocytes on STO feeder cells. In Vitro Cell. Dev. Biol. 30A:851–858; 1994b.

    CAS  Google Scholar 

  • Tablot, N. C.; Paape, M. J. Continuous culture of pig tissue-derived macrophages. Methods Cell Sci. 18:315–327; 1996.

    Article  Google Scholar 

  • Talbot, N. C.; Pursel, V.; Caperna, T. J., et al. Ultrastructure and enzymatic characteristics of the PICM-19 pluripotent fetal liver cell line. Exp. Cell Res. 225:22–34; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Tanaka, M. A histochemical study on the activity of gamma-glytamyl transpeptidase in liver disease. Acta Pathol. Jpn. 24:651–665; 1974.

    PubMed  CAS  Google Scholar 

  • Toole, B. Proteoglycans and hyaluronan in morphogenesis and differentiation. In: Hay, E. D., ed. Cell biology of the extracellular matrix. 2nd ed. New York: Plenum Press; 1991:305–341.

    Google Scholar 

  • Van Eyken, P.; Desmet, V. J. Bile duct cells. In: LeBouton, A. V., ed. Molecular and cell biology of the liver. Baton Raton, Florida: CRC Press; 1993:475–524.

    Google Scholar 

  • Vroman, B.; LaRusso, N. F. Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. Lab. Invest. 74:303–313; 1996.

    PubMed  CAS  Google Scholar 

  • Yang, L. I.; Faris, R. A.; Hicks, D. C. Long-term culture and characteristics of normal rat liver bile duct epithelial cells. Gastroenterology 104:840–852; 1993.

    PubMed  CAS  Google Scholar 

  • Yokoyama, H. O.; Wilson, M. E.; Tsuboi, K. K., et al. Regeneration of mouse liver after partial hepatectomy. Cancer Res. 13:80–85; 1953.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. USDA, ARS, LPSI, Gene Evaluation and Mapping Laboratory, Beltsville Agricultural Research Center, Building 200, Room 13, 20705, Beltsville, Maryland

    Neil C. Talbot

  2. USDA, ARS, LPSI, Growth Biology Laboratory, Beltsville Agricultural Research Center, Building 200, Room 202, 20705, Beltsville, Maryland

    Thomas J. Caperna

Authors
  1. Neil C. Talbot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas J. Caperna
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Talbot, N.C., Caperna, T.J. Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver. In Vitro Cell.Dev.Biol.-Animal 34, 785–798 (1998). https://doi.org/10.1007/s11626-998-0033-x

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-998-0033-x

Key words

Search

Navigation