EfficientIn Vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells

Abstract

Efficient transport of bile acids, a typical characteristic of hepatocytes, is partially lost in most hepatoma cell lines and in normal hepatocytes after some days in culture. We have tested whether the polarized rat hepatoma-human fibroblast hybrid WIF (hybrids between W138 and Fao cells) cells previously obtained by our group were able to perform vectorial transport of the fluorescent bile acid derivative cholylglycylamidofluorescein (CGamF) towards the bile canaliculi (BC). Four different WIF clones were analyzed. All were well polarized, as shown by the formation of spherical and even tubular BC-like structures and by the restricted localization at the BC, visualized by immunofluorescence, of the apical membrane marker HA4, a possible bile acid carrier. WIF-B and its subclone WIF-B9 were found to accumulate CGamF in 65% to 75% of their BC. This transport was time, temperature, and partly sodium dependent and was inhibited by coincubation with the parental natural bile salt cholylglycine. Dinitrophenyl glutathione, a substrate of the canalicular multispecific organic anion transporter, did not inhibit CGamF canalicular secretion, whereas it greatly impaired the canalicular secretion of a non-bile acid organic anion, fluorescein, generated intracellularly from fluorescein diacetate. Confocal microscopy confirmed the presence of CGamF in the cytoplasm, supporting a transcellular route from medium to BC. In contrast, two other polarized clones exhibited a poor ability (WIF 12-6) or no ability (WIF12-1 TG␦) to vectorially transport CGamF. In conclusion, WIF-B and WIF-B9 exhibit not only structural but also functional polarity, at least as far as vectorial organic anion transport is concerned. (HEPATOL-OGY 1998;27:576-583.)

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties

“…5 ). Similar fl uorescent conjugates of bile acids have been extensively used to study the mechanisms of uptake and excretion of bile acids by hepatocytes (37)(38)(39)(40). These bile acid fl uorescent analogs were characterized in a previous study, where DCA-NBD and UDCA-NBD were shown to have a superfi cial location in lipid membranes and to interact preferentially with less ordered liposomes, in agreement with results obtained with unmodifi ed bile acids ( 14 ).…”

WIF-B cells as a model for alcohol-induced hepatocyte injury

“…Therefore, an in vitro model that closely resembles normal hepatocytes would be beneficial for elucidating the injury-inducing mechanisms of ethanol. It has been previously established that the WIF-B cell line, a rat hepatoma/human fibroblast hybrid, has many functional and morphological similarities to hepatocytes [7][8][9][10][11][12][13]. This cell line forms functional bile canaliculi, obtains a hepatocyte-like cell polarity, expresses many hepatocytespecific genes, and is stable over many passages.…”

“…This cell line forms functional bile canaliculi, obtains a hepatocyte-like cell polarity, expresses many hepatocytespecific genes, and is stable over many passages. This cell line has been extensively used to study the effects of hepatocyte-specific polarity on intercellular communication, bile acid transport, and protein and membrane trafficking pathways [8,[10][11][12]40].…”

“…The WIF-B cell line is a highly differentiated and polarized rat hepatoma hybrid, which forms bile canaliculi-like structures [7]. This cell line has been reported as a good in vitro model for studying many hepatocyte-specific phenomena, such as hepatocyte polarity [8,9], protein secretion [7], bile acid transport [10], intercellular communication [11], and protein transport [12]. This cell line is also stable over many passages [13].…”

Small hepatocytes in culture develop polarized transporter expression and differentiation

“…Hence, the proliferative potential of mature hepatocytes originating from small hepatocytes seems to be limited in vitro, indicating the requirement of alternative strategies for the generation of high numbers and possible in vitro mass production of bile-salt-and organic-anion-transportcompetent hepatocytes. Such alternative procedures might include: (1) culturing primary hepatocytes under proliferating conditions (Block et al, 1996;Michalopoulos et al, 1999); (2) further development of partially polarized hepatoma derived cell lines such as Wif-B (Bender et al, 1999;Bravo et al, 1998) or H4IIEC3 (Ng et al, 2000) cells; and (3) isolation, expansion and differentiation of bone-marrow-derived stem cells (Avital et al, 2001;Schwartz et al, 2002). The last of these procedures is especially attractive because a subpopulation of bonemarrow-derived stem cells can acquire differentiated hepatocellular metabolic functions (Avital et al, 2001;Schwartz et al, 2002), has the capability to repopulate regenerating liver in vivo (Avital et al, 2001) and can restore certain biochemical functions of diseased liver (Lagasse et al, 2000).…”