Abstract
Few in vitro models expressing complex hepatocyte polarity are available. We used the unpolarized rat Fao cell line to isolate the polarized WIF-B line. These complex rat-human hybrid cells form functional simple bile canaliculi. To obtain Fao-derived polarized models with a simpler chromosome content and developed bile canaliculi, we employed two approaches. Partial success was achieved with monochromosomal hybrids. As shown by the immunolocalization of apical, basolateral, and tight-junctional proteins, monochromosomal hybrid 11-3 cells were polarized. They formed simple functional bile canaliculi and transiently expressed the typical polarity of simple epithelial cells. One subclone blocked in this polarity state was isolated. A more robust approach was provided by spheroid culture, a three-dimensional system that strengthens cell-cell contacts. Transient spheroid culture induced irreversible polarization of Fao cells. This induction occurred in most spheroids (approximately 1% of the cells). From populations enriched in stably polarized cells, we generated new polarized cell models, designated Can. Can 3-1 cells formed simple functional bile canaliculi when plated at high density. Regardless of plating density, Can 9 and Can 10 cells formed long tubular branched canaliculi competent for vectorial transport of organic anions and bile acids, and involving several dozen adjacent cells. Thus, we have generated new cell models stably expressing typical hepatocyte polarity. Among these models, Can 9 and Can 10 are the first capable of forming functional, highly developed bile canaliculi similar to those formed in vivo.
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Abbreviations
- BC:
-
bile canaliculi
- CGamF:
-
cholylglycylamidofluorescein
- DPPIV:
-
dipeptidylpeptidase IV
- FDA:
-
fluorescein diacetate
- PDI:
-
protein disulfide isomerase
- PBS:
-
phosphate-buffered saline
- P-gp:
-
P glycoprotein
- PM:
-
plasma membrane
- Poly-HEMA:
-
poly-(2-hydroxyethyl metacrylate)
- Rh 123:
-
rhodamine 123
- RCCS:
-
rotary cell culture system
- TER:
-
transepithelial resistance
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Acknowledgements
We thank A. Hubbard, B. R. Stevenson, and D. Keppler for kindly providing antibodies, and J.G. Marin for generously providing cholylglycylamidofluorescein. We are grateful to L. Sperling and P. Colosetti for careful reading of the manuscript.
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This work was supported by grants from the Association pour la Recherche sur le Cancer (no.6551), the Institut Curie (PIC Signalisation Cellulaire, no. 914) and the Institut National de la Santé et de la Recherche Médicale (contract PRISME 98-09).
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Peng, X., Grosse, B., Le Tiec, B. et al. How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi. Cell Tissue Res 323, 233–243 (2006). https://doi.org/10.1007/s00441-005-0067-2
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DOI: https://doi.org/10.1007/s00441-005-0067-2