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

Benjamin Vroman, Nicholas F La Russo

Research output: Contribution to journalArticle

107 Citations (Scopus)

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. γ-glutamyl-transpeptidase (γGT) 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 epical or basolateral chambers. When seeded with approximately 1 x 105 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 epical 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, γ-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.

Original languageEnglish (US)
Pages (from-to)303-313
Number of pages11
JournalLaboratory Investigation
Volume74
Issue number1
StatePublished - Jan 1996

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Intrahepatic Bile Ducts
Epithelial Cells
Keratin-7
Keratin-19
Scanning Transmission Electron Microscopy
Desmin
gamma-Glutamyltransferase
Cilia
Tight Junctions
Vimentin
Colforsin
Somatostatin
Electric Impedance
Cyclic AMP
Collagen
Somatostatin Receptors
Microvilli
Bile
Cultured Cells
Immunohistochemistry

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. / Vroman, Benjamin; La Russo, Nicholas F.

In: Laboratory Investigation, Vol. 74, No. 1, 01.1996, p. 303-313.

Research output: Contribution to journalArticle

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