Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration
Abstract
The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca 2+ -free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca 2+ -free buffer. The cumulative uptake of [ 3 H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca 2+ -free buffer, due to accumulation of taurocholate in canalicular spaces. When [ 3 H]taurocholate was preloaded in the day 5sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca 2+ -free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ∼8% at day 0 to ∼60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.
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Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes
Marion, T. L., Perry, C. H., Claire, R. L. S., & Brouwer, K. L. R. (2012) · Toxicology and Applied Pharmacology · doi:10.1016/j.taap.2012.02.002 · Cited by 59 · see citing works
“…membrane domains and are functional over days in culture, and over time adjacent cells form intact, bile-canaliculi-like structures sealed by tight junctions into which endogenous and exogenous compounds may be secreted (LeCluyse et al. 1994; Liu et al. 1999a; Liu et al. 1999b; Liu et al. 1999c).”
BackgroundMethodSelf-citationSwift, B., Nebot, N., Lee, J. K., et al. (2013) · Drug Metabolism and Disposition · doi:10.1124/dmd.112.048181 · Cited by 59 · see citing works
Self-citationLee, J. K., Marion, T. L., Abe, K., Lim, C., Pollock, G. M., & Brouwer, K. L. R. (2009) · Journal of Pharmacology and Experimental Therapeutics · doi:10.1124/jpet.109.156653 · Cited by 57 · see citing works
“…hepatocyte culture because the cells repolarize, regain their proper morphology, and maintain liver-specific metabolic activity, including stable expression of cytochrome P450 enzymes over longer times (Liu et al., 1998, 1999a,b,c; Hamilton et al., 2001; Hewitt et al., 2007; Mingoia et al., 2007).”
BackgroundMethodInfluentialSelf-citationJemnitz, K., Veres, Z., & Vereczkey, L. (2010) · Toxicological Sciences · doi:10.1093/toxsci/kfq044 · Cited by 57 · see citing works
“Human and rat SCH are commonly used to estimate the biliary efflux of compounds and also to assess hepatotoxic potential of drugs due to interaction with biliary efflux processes (Kemp and Brouwer 2004; Liu et al., 1999).”
BackgroundMohamed, L. A., Qosa, H., & Kaddoumi, A. (2015) · ACS Chemical Neuroscience · doi:10.1021/acschemneuro.5b00040 · Cited by 58 · see citing works
Kimura, H., Nishikawa, M., Kutsuzawa, N., Tokito, F., Kobayashi, T., Kurniawan, D. A., Shioda, H., Cao, W., Shinha, K., Nakamura, H., et al. (2024) · Drug Metabolism and Pharmacokinetics · doi:10.1016/j.dmpk.2024.101046 · Cited by 61 · see citing works
Tchaparian, E. H., Houghton, J. S., Uyeda, C., Grillo, M. P., & Jin, L. (2011) · Drug Metabolism and Disposition · doi:10.1124/dmd.111.039545 · Cited by 55 · see citing works
“They are extensively used as an experimental model to investigate metabolism, transport, and hepatotoxicity of investigational drugs (Liu et al., 1999; Ansede et al., 2010).”
BackgroundShen, D., Liang, X., Gawinowicz, M. A., & Gottesman, M. M. (2004) · PubMed · doi:10.1124/mol.66.4 · Cited by 31 · see citing works
“Well preserved liver-specific functions in sandwich-cultured rat hepatocytes make this a valuable in vitro model to study the expression and function of transport proteins, and predict in vivo hepatobiliary clearance (Liu et al., 1999a).”
BackgroundMethodInfluentialSelf-citationSlizgi, J. R., Lu, Y., Brouwer, K. R., St. Claire, R. L., Freeman, K. M., Pan, M., Brock, W. J., & Brouwer, K. L. R. (2016) · Toxicological Sciences · doi:10.1093/toxsci/kfv231 · Cited by 53 · see citing works
“The biliary excretion index (BEI) was calculated according to the following equation (Liu et al., 1999a): BEIð%Þ ¼ AccumulationStandard HBSS AccumulationCa2þ free HBSS AccumulationStandard HBSS 100 The in vitro biliary clearance (Clbiliary) was determined using the following equation (Ghibellini et…”
MethodSelf-citationTurncliff, R. Z., Tian, X., & Brouwer, K. L. R. (2006) · Biochemical Pharmacology · doi:10.1016/j.bcp.2006.02.004 · Cited by 53 · see citing works
Self-citationRegulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver
da Cruz, L. N., Guerra, M. T., Kruglov, E. A., Mennone, A., Garcia, C. R. S., Chen, J., & Nathanson, M. H. (2010) · Hepatology · doi:10.1002/hep.23625 · Cited by 53 · see citing works
“This cell system was used because it preserves structural and functional polarity of hepatocytes.(37,38) CMFDA accumulated in the canalicular lumen of hepatocytes (Fig.”
MethodBile canalicular dynamics in hepatocyte sandwich cultures
Reif, R., Karlsson, J., Günther, G., Beattie, L., Wrangborg, D., Hammad, S., Begher‐Tibbe, B., Damle‐Vartak, A., Melega, S., Kaye, P. M., et al. (2015) · Archives of Toxicology · doi:10.1007/s00204-015-1575-9 · Cited by 50 · see citing works
“Assays to measure hepatobiliary excretion of bile acids and chemical compounds have been established (Bi et al. 2006; Kostrubsky et al. 2003; Liu et al. 1999c).”
BackgroundBruyn, T. D., Sempels, W., Snoeys, J., et al. (2014) · Journal of Pharmaceutical Sciences · doi:10.1002/jps.23933 · Cited by 52 · see citing works
Self-citationCastell, J. V., & Gómez-Lechón, M. J. (2008) · Methods in molecular biology · doi:10.1007/978-1-59745-201-4_4 · Cited by 51 · see citing works
Vildhede, A. M., Mateus, A., Khan, E. K., et al. (2016) · Drug Metabolism and Disposition · doi:10.1124/dmd.115.066746 · Cited by 51 · see citing works
“Time-dependent disposition of pitavastatin in SCHHs was assessed using a method adapted from the originators to the B-CLEAR technology (Qualyst Transporter Solutions, Research Triangle Park, NC) (Liu et al., 1999).”
MethodLu, C., Berg, C., Prakash, S. R., et al. (2008) · Drug Metabolism and Disposition · doi:10.1124/dmd.107.019000 · Cited by 51 · see citing works
Kim, Y.-H., Larkin, A. L., Davis, R. M., et al. (2010) · Tissue Engineering Part A · doi:10.1089/ten.tea.2009.0695 · Cited by 49 · see citing works
“35,36 The presence of canaliculi was determined through the localization of a fluorescent di-peptyl peptidase IV enzyme.”
Hoffmaster, K. A., Zamek‐Gliszczynski, M. J., Pollack, G. M., et al. (2004) · Drug Metabolism and Disposition · doi:10.1124/dmd.104.001420 · Cited by 46 · see citing works
Self-citationYue, W., Abe, K., & Brouwer, K. L. R. (2008) · Molecular Pharmaceutics · doi:10.1021/mp800100e · Cited by 45 · see citing works
Self-citationLI, X., Su, M., WEST, C. A., He, C., Swanson, S., Secomb, T. W., & Mentzer, S. J. (2001) · In Vitro Cellular & Developmental Biology - Animal · doi:10.1290/1071-2690(2001)037 · Cited by 17 · see citing works
Self-citationChatterjee, S., Bijsmans, I. T. G. W., van Mil, S. W. C., Augustijns, P., & Annaert, P. (2013) · Toxicology in Vitro · doi:10.1016/j.tiv.2013.10.020 · Cited by 44 · see citing works
Role of AMP-activated protein kinase α1 in 17α-ethinylestradiol-induced cholestasis in rats
Li, X., Liu, R., Luo, L., Yu, L., Chen, X., Sun, L., Wang, T., Hylemon, P. B., Zhou, H., Jiang, Z., et al. (2016) · Archives of Toxicology · doi:10.1007/s00204-016-1697-8 · Cited by 44 · see citing works
“Rat primary hepatocytes were isolated by a twostep collagenase perfusion method and cultured in a collagen-sandwich configuration as reported previously (Liu et al. 1999).”
MethodInvolvement of Organic Cation Transporters in the Kinetics of Trimethylamine N-oxide
Miyake, T., Mizuno, T., Mochizuki, T., Kimura, M., Matsuki, S., Irie, S., Ieiri, I., Maeda, K., & Kusuhara, H. (2017) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2017.04.067 · Cited by 44 · see citing works
Chandra, P., LeCluyse, E. L., & Brouwer, K. L. R. (2001) · In Vitro Cellular & Developmental Biology - Animal · doi:10.1007/bf02577575 · Cited by 44 · see citing works
“This polarized excretion confirms that intact canalicular networks were formed in the sandwich-cultured hepatocytes (Liu et al., 1999b).”
BackgroundMethodInfluentialSelf-citationde Haan, L. R., Reiniers, M. J., Reeskamp, L. F., Belkouz, A., Ao, L., Cheng, S., Ding, B., van Golen, R. F., & Heger, M. (2022) · Antioxidants · doi:10.3390/antiox11081424 · Cited by 45 · see citing works
“DCFH2-DA is converted to DCF at a higher rate in DMEM compared to RPMI 1640 medium [31,68], so the primary hepatocytes were exposed to more DCF and less DCFH2-DA than the HepG2 cells.”
MethodInfluentialWolf, K., Vora, S., Webster, L. O., Generaux, G. T., Polli, J. W., & Brouwer, K. L. R. (2009) · Toxicology in Vitro · doi:10.1016/j.tiv.2009.08.009 · Cited by 43 · see citing works
“…IH -PA Author M anuscript N IH -PA Author M anuscript min/kg) of taurocholate were calculated using B-CLEAR® technology (Qualyst, Inc., Raleigh, NC; Liu et al., 1999a) and the following equations: (1) (2) where substrate accumulation in cells+bile was determined in hepatocytes preincubated in…”
BackgroundMethodInfluentialSelf-citationYamasaki, C., Ishida, Y., Yanagi, A., Yoshizane, Y., Kojima, Y., Ogawa, Y., Kageyama, Y., Iwasaki, Y., Ishida, S., Chayama, K., et al. (2020) · PLoS ONE · doi:10.1371/journal.pone.0237809 · Cited by 42 · see citing works
“This observation indicated that in the presence of Ca the integrity of the bile canalicular networks remains intact, while in the absence of Ca2 the integrity of the canalicular space is disrupted, causing leakage of the canalicular contents, as reported previously [29].”
ResultTreyer, A., & Müsch, A. (2013) · Comprehensive physiology · doi:10.1002/j.2040-4603.2013.tb00490.x · Cited by 43 · see citing works
Kimoto, E., Walsky, R. L., Zhang, H., Bi, Y., Whalen, K. M., Yang, Y.-S., Linder, C. D., Xiao, Y., Iseki, K., Fenner, K. S., et al. (2011) · Drug Metabolism and Disposition · doi:10.1124/dmd.111.039297 · Cited by 42 · see citing works
“The initial uptake rate was calculated from the slope between 0.5 and 5 min, whereas biliary clearance was estimated as previously reported (Liu et al., 1999b).”
BackgroundMethodInfluentialWang, S., Li, X., Guo, H., et al. (2016) · Hepatology Research · doi:10.1111/hepr.12788 · Cited by 42 · see citing works
Jemnitz, K., Veres, Z., Tugyi, R., & Vereczkey, L. (2009) · Toxicology in Vitro · doi:10.1016/j.tiv.2009.10.009 · Cited by 42 · see citing works
Novel Bile Acid-Dependent Mechanisms of Hepatotoxicity Associated with Tyrosine Kinase Inhibitors
Saran, C., Sundqvist, L., Ho, H. S. S., Niskanen, J., Honkakoski, P., & Brouwer, K. L. R. (2021) · Journal of Pharmacology and Experimental Therapeutics · doi:10.1124/jpet.121.000828 · Cited by 41 · see citing works
“Following a 24-hour exposure to TKIs (day 5), the biliary excretion index was measured using B-CLEAR technology (Brouwer et al., 2013; Liu et al., 1999; Swift et al., 2010).”
MethodSelf-citationTetsuka, K., Ohbuchi, M., & Tabata, K. (2017) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2017.05.010 · Cited by 41 · see citing works
Barber, J., Stahl, S. H., Summers, C., Barrett, G., Park, B. K., Foster, J. R., & Kenna, J. G. (2015) · Toxicological Sciences · doi:10.1093/toxsci/kfv159 · Cited by 39 · see citing works
Heparin hydrogel sandwich cultures of primary hepatocytes
Foster, E. E., You, J., Siltanen, C. A., Patel, D., Haque, A., Anderson, L., & Revzin, A. (2015) · European Polymer Journal · doi:10.1016/j.eurpolymj.2014.12.033 · Cited by 38 · see citing works
Nakakariya, M., Ono, M., Amano, N., et al. (2011) · Drug Metabolism and Disposition · doi:10.1124/dmd.111.042101 · Cited by 38 · see citing works
“…the biliary clearance of some drugs, such as angiotensin II receptor blockers, HMG-CoA reductase inhibitors, and -lactam antibiotics, showed correlation between sandwich-cultured rat or human hepatocytes and in vivo (Liu et al., 1999a; Abe et al., 2008, 2009; Fukuda et al., 2008; Li et al., 2010).”
BackgroundMethodInfluentialCharacterization of Chemically Induced Hepatotoxicity in Collagen Sandwiches of Rat Hepatocytes
Farkas, D., & Tannenbaum, S. R. (2005) · Toxicological Sciences · doi:10.1093/toxsci/kfi145 · Cited by 37 · see citing works
Vée, M. L., Jouan, E., Stieger, B., et al. (2015) · PLoS ONE · doi:10.1371/journal.pone.0121232 · Cited by 36 · see citing works
“Biliary excretion index (BEI) reflecting canalicular secretion of taurocholate was calculated using the following equation [47]:”
MethodSelf-citationConstrained spheroids for prolonged hepatocyte culture
Tong, W. H., Fang, Y., Yan, J., Hong, X., Singh, N. H., Wang, S. R., Nugraha, B., Xia, L., Fong, E. L. S., Iliescu, C., et al. (2015) · Biomaterials · doi:10.1016/j.biomaterials.2015.11.036 · Cited by 36 · see citing works
“For example, the current in vitro gold standard, the collagen sandwich culture system, has been shown to be capable of reestablishing and maintaining polarity in primary rat hepatocytes for up to 5 days [58, 69].”
BackgroundViability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure
Kemp, D. C., & Brouwer, K. L. R. (2004) · Toxicology in Vitro · doi:10.1016/j.tiv.2004.04.014 · Cited by 36 · see citing works
Self-citationYe, Z., Camus, S., Augustijns, P., & Annaert, P. (2010) · Biopharmaceutics & Drug Disposition · doi:10.1002/bdd.701 · Cited by 36 · see citing works
Retention of Transporter Activities in Cryopreserved, Isolated Rat Hepatocytes
Houle, R., Raoul, J., Lévesque, J.-F., et al. (2003) · Drug Metabolism and Disposition · doi:10.1124/dmd.31.4.447 · Cited by 35 · see citing works
“In this model the billiary efflux transporters such as MRP2 are maintained, however, uptake transporters such as NTCP are significantly down-regulated (Liu et al., 1999).”
BackgroundLei, X., Arooz, T., Zhang, S., Tuo, X., Xiao, G., Susanto, T. A. K., Sundararajan, J., Cheng, T., Kang, Y., Poh, H. J., et al. (2012) · Biomaterials · doi:10.1016/j.biomaterials.2012.06.078 · Cited by 35 · see citing works
Yin, J. B., Meng, Q., Zhang, G., & Sun, Y. (2009) · Chemico-Biological Interactions · doi:10.1016/j.cbi.2009.04.004 · Cited by 35 · see citing works
Cheng, Y., Woolf, T. F., Gan, J., & He, K. (2015) · Chemico-Biological Interactions · doi:10.1016/j.cbi.2015.11.029 · Cited by 34 · see citing works
Sharma, S., Ellis, E. C. S., Gramignoli, R., Dorko, K., Tahan, V., Hansel, M. C., Mattison, D. R., Caritis, S. N., Hines, R. N., Venkataramanan, R., et al. (2012) · Drug Metabolism and Disposition · doi:10.1124/dmd.112.044891 · Cited by 34 · see citing works
“A large BEI (as high as taurocholate) indicates extensive excretion of substrate into the canalicular space via transporters (Liu et al., 1999).”
BackgroundMethodInfluentialTurncliff, R., Hoffmaster, K. A., Kalvass, J., Pollack, G. M., & Brouwer, K. (2006) · Journal of Pharmacology and Experimental Therapeutics · doi:10.1124/jpet.106.102616 · Cited by 34 · see citing works
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Primary hepatocyte cultures as prominentin vitrotools to study hepatic drug transporters
Ramboer, E., Vanhaecke, T., Rogiers, V., & Vinken, M. (2013) · Drug Metabolism Reviews · doi:10.3109/03602532.2012.756010 · Cited by 32 · see citing works
- Authors
- Liu, X., LeCluyse, E. L., Brouwer, K. R., Gan, L.-S. L., Lemasters, J. J., Stieger, B., Meier, P. J., & Brouwer, K. L. R.
- Affiliations
- Division of Drug Delivery and Disposition, School of Pharmacy, and
- Division of Bioanalysis and Drug Metabolism, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709; and
- University of North Carolina at Chapel Hill (US)
- University Hospital Zurich (CH)
- Venue
- American Journal of Physiology-Gastrointestinal and Liver Physiology
- Publisher
- American Physiological Society
- Published
- 1999-07-01
- Volume
- 277
- Issue
- 1
- Pages
- G12-G21
- ISSN
- 0193-1857 (ISSN-L)
- 1522-1547
- OpenAlex
- W2101528448
- Semantic Scholar
- 501c1cfc2938afc5aa6a1bdf0351de034d80a0bc
- Cited by
- 226
- References
- 30
- Citations by year
- 2026: 2 · 2025: 5 · 2024: 5 · 2023: 2 · 2022: 6 · 2021: 7 · 2020: 4 · 2019: 5 · 2018: 5 · 2017: 8 · 2016: 9 · 2015: 16 · 2014: 12 · 2013: 21 · 2012: 11
- FWCI
- 1.56
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- American Journal of Physiology-Gastrointestinal and Liver Physiology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM041935, GM 41935)
- Topics
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Keywords
- biliary excretion, hepatocyte polarity, MRP2, taurocholate, bile canaliculi, organic anion transport, bile acid efflux
- MeSH terms
- Animals, Bile, Carrier Proteins, Cell Membrane, Cells, Cultured, Collagen, Cytological Techniques, Fluoresceins, Liver, Male, Taurocholic Acid, Time Factors, Immunoblotting, Cell Polarity, Rats, Wistar, Anion Transport Proteins, Rats
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