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

Talbot, Neil C.; Caperna, Thomas J.

doi:10.1007/s11626-998-0033-x

Cited by 18 publications

(16 citation statements)

References 33 publications

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“…The cyclic nucleotide analogs, dbcAMP (1 or 2 mM) and dbcGMP (2 mM), produced relatively weak or no responses, respectively, after 2-3 h (table 1). A similar response to forskolin was found in pig liver-derived bile duct tissue cultures [Talbot and Caperna, 1998] which were tested as a comparative control (table 4).…”

Section: Induction Of Lumen Fluid Transport By Camp Inducers Bioactisupporting

confidence: 61%

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The PICM-19 Cell Line as an in vitro Model of Liver Bile Ductules: Effects of cAMP Inducers, Biopeptides and pH

Talbot¹,

Caperna²,

Wells³

2002

Cells Tissues Organs

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The PICM-19 fetal liver cell line was isolated from the primary culture and spontaneous differentiation of pig epiblast cells, i.e. embryonic stem cells. PICM-19 cells were induced to differentiate into mostly ductular formations by culturing at pH 7.6–7.8. The ductules were functionally assayed by treatment with cAMP inducing agents and bioactive peptides reported to influence the secretory activity of liver bile ductules. The secretory response of the cells was assessed by qualitative or quantitative measurement of the cross-sectional area of the ductal lumens and the appearance of biliary canaliculi in between PICM-19 cells that had formed monolayers instead of ducts. Forskolin (10 µM) and 8-bromoadenosine 3′:5′-cyclic monophosphate (bcAMP; 2 mM) stimulated fluid transport and expansion of ductal structures in 15–20 min and stimulated the appearance and expansion of biliary canaliculi in 30–60 min. Cholera toxin (50 ng/ml) stimulates fluid transport in both ductules and canaliculi in 1–2 h, while 8-bromoguanosine 3′:5′-cyclic monophosphate (bcGMP; 2 mM) stimulated only biliary canaliculi in 2 h. Glucagon (1.4 nM) produced a similar response in 5–10 min in ductal structures only, but the response was transitory and was almost completely reversed within 30 min. Secretin (100 pM) and vasoactive intestinal peptide (75 pM) produced a sustained response with maximal ductal lumen expansion occurring in 5–10 min and neither had an immediate effect on canaliculi. Somatostatin (0.5 µM) and gastrin (1 µM) caused marked reduction or disappearance of ductal lumens in 30–60 min, but was ineffective in reversing secretin (100 nM)-induced duct distension. Application of the adrenergic agonists, epinephrine, isoproterenol, and phenylephrine (100 µM), resulted in the complete shrinkage of ductal lumens in 20–30 min. A shift to pH 7.0–7.2 resulted in almost complete reduction of ductal lumens, while a shift to pH 7.8–8.0 resulted in expansion, although not full expansion, of the ductal lumens. PICM-19 bile duct cultures were positive for cytokeratin-7, aquaporin-1 and aquaporin-9 by Western blot analysis. The amounts of these proteins increased in the cultures as differentiation proceeded over time. Transmission electron microscopy revealed that the ductal structures were usually sandwiched between SIM mouse, thioguanine- and ouabain-resistant (STO) feeder cells that had produced a collagen matrix. Also, the ductular PICM-19 cells possessed cilia, probably occurring as a single cilium in each cell, that projected into the lumens of the ducts. The results indicated that the in vitro-produced ductal structures of the PICM-19 cell line are a functional model for biliary epithelium.

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Section: Induction Of Lumen Fluid Transport By Camp Inducers Bioactisupporting

confidence: 61%

“…without passage of the cells and with refeeding every 3-4 days. Cultures of nearly pure pig bile duct epithelium derived from adult pig liver tissue were used for comparative purposes in some assays, and they were grown and maintained on STO feeder cells as previously described [Talbot and Caperna, 1998]. …”

Section: Cell Culture and Reagentsmentioning

confidence: 99%

The PICM-19 Cell Line as an in vitro Model of Liver Bile Ductules: Effects of cAMP Inducers, Biopeptides and pH

Talbot¹,

Caperna²,

Wells³

2002

Cells Tissues Organs

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“…Information about AQP expression in the pig liver will provide a basis with which to assess the functional validity of in vitro models of pig hepatocytes and pig bile duct cells [Talbot et al, 1996;Talbot and Caperna, 1998]. Recently, we have found that pig bile ductules, grown and differentiated in vitro, rapidly transport fluid into their lumens after exposure to physiological levels of secretin or to inducers of cAMP [Talbot et al, 2002].…”

Section: Discussionmentioning

confidence: 99%

“…Our laboratory is involved with developing in vitro models of embryonic and adult pig liver and biliary epithelium [Talbot et al, 1996;Talbot and Caperna, 1998]. The functional validity of these in vitro models is in part dependent on knowledge of normal pig hepatocyte and bile duct epithelium protein expression [Talbot et al, 2003].…”

Section: Introductionmentioning

confidence: 99%

Analysis of the Expression of Aquaporin-1 and Aquaporin-9 in Pig Liver Tissue: Comparison with Rat Liver Tissue

Talbot¹,

Garrett²,

Caperna

2003

Cells Tissues Organs

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Aquaporins (AQPs) are cellular proteins involved with the movement of water across cell membranes and are fundamentally important to the fluid transport in the bile ducts and ductules of the liver. An immunohistochemical analysis of AQP-1 and AQP-9 was undertaken to describe their expression in fetal and adult pig liver, while immunoreagents specific to some other AQPs were screened for their efficacy on pig liver tissues. Anti-AQP-1 antibody reacted with the bile duct of the portal space and the bile ductules at the periphery of the liver lobules. Histological identification of bile ductules was confirmed by positive reactivity with anti-cytokeratin-7 and antilaminin immunostaining. Anti-AQP-1 signals were also pronounced in the endothelium of the portal space blood vessels and peripheral distributing venules. Antibody to AQP-9 reacted strongly with small ductules peripheral to the liver lobules, but only weakly with the bile ducts of the portal space. Anti-AQP-adipose antibody bound to the smooth muscle cells of the arteries in the portal space and sporadically with certain binucleated cells in the liver lobule. Antibodies to AQP-3, AQP-4, AQP-7, and AQP-8 were nonreactive with any of the tissues of the adult pig liver. For comparative purposes, immunohistochemical analysis of rat liver tissue was done with the anti-AQP-1 and AQP-9 antibodies. Anti-AQP-1 reacted weakly with the rat liver’s bile ducts, but robustly with the endothelium of the liver’s veins and arteries. It also reacted strongly with the central vein of the rat liver lobules, and, because the staining was continuous with hepatic sinusoids, it appeared that the reactivity was specific to the endothelial cells. Anti-AQP-9 antibodies reacted with rat hepatocytes and was not associated with the canaliculi, as judged by concurrent phalloidin staining of actin. The results indicate that specific AQPs are expressed in the tissues of the pig liver and that AQP-9 expression is distinct from its expression in the rat liver.

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“…They also secreted alpha-fetoprotein, albumin, and other liver-specific proteins (Talbot et al 1994a. Primary cultures of fetal pig liver tissue (Talbot et al 1994b;Talbot and Caperna 1998) were similar to PICM-19 cells in differentiation potential, morphology, and protein/ enzyme expression. The in vivo-like responses of the PICM-19 ductules to secretin and cAMP inducers (Talbot et al 2002) were similar to that of ductules formed in vitro by finite porcine cholangiocyte cell lines established from adult pig liver (Talbot and Caperna 1998).…”

Section: Introductionmentioning

confidence: 88%

Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays

Willard

Shappell²,

Meekin³

et al. 2009

In Vitro Cell.Dev.Biol.-Animal

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Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM- 19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with β-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6β-OH-, 2α-OH- and 2β-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD50 was calculated to be 14.9±0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrenetreated cultures and untreated controls; CD50 were 1.59 μM and 31 μM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.

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

Cited by 18 publications

References 33 publications

The PICM-19 Cell Line as an in vitro Model of Liver Bile Ductules: Effects of cAMP Inducers, Biopeptides and pH

The PICM-19 Cell Line as an in vitro Model of Liver Bile Ductules: Effects of cAMP Inducers, Biopeptides and pH

Analysis of the Expression of Aquaporin-1 and Aquaporin-9 in Pig Liver Tissue: Comparison with Rat Liver Tissue

Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays

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