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Lab Invest. 1996 Jan;74(1):303-13.

Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.

Author information

1
Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.

Abstract

The study of intrahepatic bile duct epithelial cells (i.e., cholangiocytes) has been limited by the lack of a polarized in vitro model that allows easy access to both apical and basolateral cell surfaces. Therefore, we developed a cell line of polarized normal rat cholangiocytes (NRCs) and established conditions that produced a confluent monolayer of cells grown on collagen-coated filters of tissue culture inserts. We passaged NRCs at high density to collagen-coated, tissue-culture inserts and measured transepithelial electrical resistance. We evaluated ultrastructural features by transmission and scanning electron microscopy. gamma-glutamyl-transpeptidase (gamma GT) was visualized in cultured cells by enzyme histochemistry, and cytokeratin (CK)-7, CK-19, vimentin, and desmin staining was done by immunohistochemistry. We studied the biologic responsiveness and functional polarity of NRCs by measuring their levels of cyclic AMP after addition of forskolin with or without somatostatin to either the apical or basolateral chambers. When seeded with approximately 1 x 10(5) cells/cm2, the NRCs formed a confluent monolayer in 72 hr. Transepithelial electrical resistance increased over time, achieving a maximum of 625 (+- 25) ohms.cm2 by 1 week after confluence. Transmission and electron microscopy scanning showed the apical cell surface to be tightly packed with microvilli with a heterogeneous display of cilia ranging from none to 20 to 30 cilia/cell. On transmission, apically positioned tight junctions and vesicles were apparent; nuclei were oriented basally and the basolateral surface was characterized by membrane interdigitations. NRCs stained positively for the cholangiocyte marker proteins, gamma-glutamyl-transpeptidase, CK-7, and CK-19, and negative for the mesenchymal markers, vimentin, and desmin. Exposure of the basolateral (but not the apical) cell surface to somatostatin caused a 60% inhibition of forskolin-induced increases in intracellular levels of cyclic AMP, suggesting the presence of somatostatin receptors exclusively on the basolateral plasma membrane domain. We have developed a unique model of primary cultures of normal rat cholangiocytes in which the apical and basolateral surfaces are easily accessible; the cells develop intermediate-strength tight junctions, retain their cholangiocyte phenotype, display morphologic and functional polarity, and are responsive to hormones. This model should be useful for the assessment of vectorial transport of solutes and other constituents of blood and bile, as well as for studying growth regulation of cholangiocytes.

PMID:
8569194
[Indexed for MEDLINE]

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