Cited by 226Drug Transport and Resistance MechanismsLiver physiology and pathologyDrug-Induced Hepatotoxicity and Protection

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Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration

Liu, Xingrong, LeCluyse, Edward L., Brouwer, Kenneth R., Gan, Liang-Sheng L., Lemasters, John J., Stieger, Bruno, Meier, Peter J., Brouwer, Kim L. R.·1999·American Journal of Physiology-Gastrointestinal and Liver Physiology

DOI 10.1152/ajpgi.1999.277.1.g12

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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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Vée, M. L., Moreau, A., Jouan, E., Denizot, C., Parmentier, Y., & Fardel, O. (2022) · Biopharmaceutics & Drug Disposition · doi:10.1002/bdd.2333 · Cited by 8
Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS
Mardal, M., Annaert, P., Noble, C., Oorts, M., & Línnet, K. (2017) · Journal of Pharmaceutical and Biomedical Analysis · doi:10.1016/j.jpba.2017.11.027 · Cited by 8
Hepatocellular Exposure of Troglitazone Metabolites in Rat Sandwich-Cultured Hepatocytes Lacking Bcrp and Mrp2: Interplay between Formation and Excretion
Yang, K., & Brouwer, K. L. R. (2014) · Drug Metabolism and Disposition · doi:10.1124/dmd.114.057190 · Cited by 8
“…within the bile canaliculi, was calculated using BCLEAR® technology (Qualyst Transporter Solutions, Durham, NC) according to the following equation (Liu et al., 1999): BEI ð%Þ ¼ AccumulationStandard HBSS 2AccumulationCa2þ 2 free HBSS AccumulationStandard HBSS 100 ð1Þ Effects of viral infection…”
MethodInfluentialSelf-citation
Photophysical characterization and flow cytometry applications of cholylamidofluorescein, a fluorescent bile acid scaffold
Roháčová, J., Marin, M. L., Martínez‐Romero, A., et al. (2008) · Photochemical & Photobiological Sciences · doi:10.1039/b806366d · Cited by 8
Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions
Kang, H. E., Malinen, M. M., Saran, C., et al. (2019) · Drug Metabolism and Disposition · doi:10.1124/dmd.119.087676 · Cited by 7
“The decreased amount of CDF retained within canaliculi-like structures in cultures treated with Ca 2+ -free buffer compared to cultures treated with standard (Ca 2+ -containing) buffer resembled the findings in primary hepatocytes (Liu et al., 1999; Pfeifer et al., 2013) and suggested that the BEI may be estimated in…”
MethodResultSelf-citation
Tacrine Sinusoidal Uptake and Biliary Excretion in Sandwich-Cultured Primary Rat Hepatocytes
Kaddoumi, A., & Mohamed, L. A. (2014) · Journal of Pharmacy & Pharmaceutical Sciences · doi:10.18433/j3801t · Cited by 7
“into 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDF), a fluorescent metabolite, which is actively effluxed into the canalicular spaces via MRP2 (16).”
BackgroundMethod
Automated Applications of Sandwich-Cultured Hepatocytes in the Evaluation of Hepatic Drug Transport
Perry, C. H., Smith, W. R., Claire, R. L. S., et al. (2011) · SLAS DISCOVERY · doi:10.1177/1087057111400192 · Cited by 7
“Hepatocytes cultured in a sandwich configuration form extensive, functional bile canalicular networks and maintain the expression, localization, and function of uptake and efflux transporters relative to hepatocytes in vivo.(2) These canalicular networks, or bile pockets, are isolated from the media by tight junctions…”
BackgroundSelf-citation
EUFEPS conference on Drug Transporters at Copenhagen: Integrative approaches in ADME research
Meijer, D. K. F., & Lennernäs, H. (2005) · European Journal of Pharmaceutical Sciences · doi:10.1016/j.ejps.2005.07.001 · Cited by 7
The influence of the choice of digestion enzyme used to prepare rat hepatocytes on xenobiotic uptake and efflux
Sinclair, J. A., Henderson, C. J., Tettey, J. N. A., et al. (2012) · Toxicology in Vitro · doi:10.1016/j.tiv.2012.07.012 · Cited by 6
NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis
Salas, G., Litta, A. A., Medeot, A. C., Schuck, V. S., Andermatten, R. B., Miszczuk, G. S., Ciriaci, N., Razori, M. V., Barosso, I. R., Pozzi, E. J. S., et al. (2024) · Biochimie · doi:10.1016/j.biochi.2024.04.002 · Cited by 6
Characterization of a Rat Multi-Cell Type 3D-Liver Microtissue System
Odermatt, A. (2013) · Journal of Tissue Science & Engineering · doi:10.4172/2157-7552.1000130 · Cited by 6
“Hepatocytes cultured as monolayers rapidly lose the expression and functional activities of key cytochrome P450 enzymes, multi-drug resistance proteins, proteins required for albumin and transferring, and glucocorticoid regeneration within the first 24-48 h [43-45].”
Background
Apical Localization of Inositol 1,4,5-Trisphosphate Receptors Is Independent of Extended Synaptotagmins in Hepatocytes
Amaya, M. J., de Oliveira, A. G., Schroeder, L. K., Allgeyer, E. S., Bewersdorf, J., & Nathanson, M. H. (2014) · PLoS ONE · doi:10.1371/journal.pone.0114043 · Cited by 6
“We observed that E-Syt1 and E-Syt2 mRNA expression is maintained after 96 hrs in culture (Figure 2a-b), when cells reach full polarity [33, 34].”
BackgroundMethod
Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes
Treijtel, N., van Eijkeren, J. C. H., Nijmeijer, S. M., der Sandt, I. C. J. D. G.-V., & Freidig, A. P. (2008) · Toxicology in Vitro · doi:10.1016/j.tiv.2008.11.001 · Cited by 6
Understanding the clinical pharmacokinetics of a GABAApartial agonist by application ofin vitrotools
Sawant, A. D., Miller, E. L., Duignan, D. B., Brodfuehrer, J., Winter, S. M., Nelson, F. R., Kamel, A. M., Obach, R. S., & Vaz, A. D. N. (2010) · Xenobiotica · doi:10.3109/00498254.2010.481057 · Cited by 6
“The biliary excretion index (BEI) was determined using B-CLEAR technology (Qualyst, Inc., Raleigh, NC) and was calculated using the equation of Liu et al (1999b) shown below: BEI Accumulation Ca Mg Accumulation Ca Mg free A 2 2 2 2 = + − − + + + + ( ) ( ) ccumulation Ca Mg2 2+ + +( )…”
Method
Bile canalicular secretion – tales from Vienna and Yale
Boyer, J. L. (2008) · Wiener Medizinische Wochenschrift · doi:10.1007/s10354-008-0591-2 · Cited by 5
“For this reason and others we have explored alternative models including WIF-B cells [8, 18] or isolated rat hepatocytes cultured in a collagen sandwich [7, 19].”
Background
Cell-based approaches for the mechanistic understanding of drug-induced cholestatic liver injury
Timor-López, E., Tolosa, L., & Donato, M. T. (2025) · Archives of Toxicology · doi:10.1007/s00204-025-04016-0 · Cited by 5
Application of monoclonal antibodies to measure metabolism of an anti‐trypanosomal compound in vitro and in vivo
Goldsmith, R. B., Gray, D. R., Yan, Z., et al. (2010) · Journal of Clinical Laboratory Analysis · doi:10.1002/jcla.20380 · Cited by 4
“Hepatocytes were isolated from male Wistar rats, and seeded onto BioCoatTM collagen plates as reported previously (12).”
Method
Recapitulating the liver niche in vitro
Yap, K. K., & Mitchell, G. M. (2022) · Advances in stem cells and their niches · doi:10.1016/bs.asn.2021.10.002 · Cited by 3
Strategic Use of Preclinical Pharmacokinetic Studies and In Vitro Models in Optimizing ADME Properties of Lead Compounds
Thakker, D. R. (2007) · Springer eBooks · doi:10.1007/978-0-387-44961-6_1 · Cited by 4
Metabolic Stability Screen in Drug Discovery
Lu, C. (2014) · Wiley eBooks · doi:10.1002/9781118541203.xen0021 · Cited by 4
Human liver cell-based assays for the prediction of hepatic bile acid efflux transporter inhibition by drugs
Fardel, O., Moreau, A., Jouan, E., Denizot, C., Vée, M. L., & Parmentier, Y. (2025) · Expert Opinion on Drug Metabolism & Toxicology · doi:10.1080/17425255.2025.2453486 · Cited by 3
“For this purpose, sandwich-cultured cells are first maintained in the presence or absence of Ca 2+ /Mg 2+ for at least 10 min; the Ca 2+ /Mg 2+ withdrawal disrupts tight junctions, which opens bile canalicular networks [117].”
Analysis of Hepatic Transport Proteins
Yang, K., Köck, K., & Brouwer, K. (2013) · AAPS advances in the pharmaceutical sciences series · doi:10.1007/978-1-4614-8229-1_9 · Cited by 3
Self-citation
The new murine hepatic 3A cell line responds to stress stimuli by activating an efficient Unfolded Protein Response (UPR)
Guantario, B., Conigliaro, A., Amicone, L., Sambuy, Y., & Bellovino, D. (2011) · Toxicology in Vitro · doi:10.1016/j.tiv.2011.09.020 · Cited by 3
“, 2004) or primary hepatocytes grown in a collagen sandwich (Liu et al., 1999), in which the tissue organization is maintained.”
Background
Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery
Tokito, F., Kiyofuji, M., Choi, H., Nishikawa, M., Takezawa, T., & Sakai, Y. (2024) · Journal of Bioscience and Bioengineering · doi:10.1016/j.jbiosc.2024.01.017 · Cited by 3
Novel multiple assessment of hepatocellular drug disposition in a single packaged procedure
Takahashi, R., Ichikawa, H., & Kanda, K. (2015) · Drug Metabolism and Pharmacokinetics · doi:10.1016/j.dmpk.2015.12.001 · Cited by 3
“CDF has been used as a model fluorescent compound to evaluate the excretion into bile canaliculi byMultidrug Resistant-associated Protein (MRP) 2 [10,21].”
Method
Transporter study methodologies
Lai, Y. (2013) · Elsevier eBooks · doi:10.1533/9781908818287.675 · Cited by 3
In Vitro/In Vivo Correlation for Transporters
Cvijić, S. (2018) · Springer eBooks · doi:10.1007/978-3-319-56637-5_22-1 · Cited by 3
Technical Challenges and Recent Advances of Implementing Comprehensive ADMET Tools in Drug Discovery
Wang, J., & Bell, L. (2012) · doi:10.1002/9781118180778.ch9 · Cited by 3
ATP-Binding-Cassette Transporters in Biliary Efflux and Drug-Induced Liver Injury
Pedersen, J. M. (2013) · KTH Publication Database DiVA (KTH Royal Institute of Technology) · Cited by 3
Evaluation of a human 3D multicellular hepatic spheroid model as a platform for studying hepatic transporters
Hartauer, M., Ho, H. S. S., Wan, M., Murphy, W. A., Tiley, J. B., Fallon, J. K., Bishop, C. E., & Brouwer, K. L. R. (2025) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-025-05996-z · Cited by 2
“This method enables differentia - tion between the total amount of compound in “cell + bile” and “cells only” by comparing accumulation in standard and Ca 2 ⁺-free HBSS, respectively [ 5, 6].”
InfluentialSelf-citation
AN IN VITRO FLUOROMETRIC ASSAY FOR EVALUATING FUNCTIONAL POLARITY OF HEPATOCYTE
Palakkan, A. A., Kumar, P. R. A., & Kumary, T. V. (2014) · International Journal of Bioassays · doi:10.21746/ijbio.2014.01.002 · Cited by 2
Transplant of Primary Human Hepatocytes Cocultured with Bone Marrow Stromal Cells to SCID Alb-uPA Mice
Mohajerani, S., Nourbakhsh, M., Cadili, A., Lakey, J. R. T., & Kneteman, N. M. (2010) · Cell Medicine · doi:10.3727/215517910x536627 · Cited by 2
In Vitro Characterization of Intestinal and Hepatic Transporters: MRP2
Alluri, R. V., Ward, P., Kunta, J. R., et al. (2013) · Methods in pharmacology and toxicology · doi:10.1007/978-1-62703-742-6_22 · Cited by 2
The Past, Current, and Future Application of Cryopreserved Human Hepatocytes in Drug Development
Zhuang, X., & Lu, C. (2016) · International Journal of Clinical Pharmacology & Pharmacotherapy · doi:10.15344/2456-3501/2016/120 · Cited by 2
Role of drug metabolism & disposition in discovery and development of new drugs
Joshi, N., Arya, R. K. K., Bisht, D., & Joshi, D. C. (2021) · World Journal of Pharmaceutical Sciences · doi:10.54037/wjps.2021.91207 · Cited by 2
In Vitro Techniques to Study Transporter-Based DDI
Bleasby, K., Chu, X., & Evers, R. (2009) · Springer eBooks · doi:10.1007/978-1-4419-0840-7_9 · Cited by 3
In VitroApproaches to Study Drug–Drug Interactions
Ferguson, S., & Bonzo, J. A. (2014) · Methods and principles in medicinal chemistry · doi:10.1002/9783527673261.ch18 · Cited by 2
Functions of the Liver
Renato, P., Correa, A. V., Nathanson, M. H., Wolkoff, A. W., Novikoff, P. M., Thevananther, S., Karpen, S. J., Elferink, R. P. J. O., Püschel, G. P., & Stieger, B. (2007) · Wiley eBooks · doi:10.1002/9780470691861.ch2b · Cited by 3
Self-citation
Comparative analysis of bile canaliculi formation in fresh and flask-delivered human hepatocytes from humanized mouse livers under sufficient oxygen supply
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Development of an in vitro metabolic dysfunction-associated steatohepatitis model to investigate altered drug metabolizing enzymes, transport proteins, and hepatobiliary disposition
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“E 2 17G in SCHH was measured using B-CLEAR ® technology (Swift et al., 2010; Liu et al., 1999; Ito et al., 2020).”
Self-citation
Distribution: Across Barriers
Eisenblaetter, T., Hagos, Y., Flörl, S., & Kühne, A. (2013) · Springer eBooks · doi:10.1007/978-3-642-25240-2_38 · Cited by 1
A Modified Procedure for Estimating the Impact of the Uptake on the Overall Biliary Clearance in Sandwich Culture of Rat Hepatocytes
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Highlight report: New methods for quantification of bile canalicular dynamics
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“Tests in this field are based on the release and subsequent analysis of the canalicular content (Liu et al., 1999a-c; Bi et al., 2006; Kostrubsky et al., 2003).”
Bile Salt Export Pump: Drug-Induced Liver Injury and Assessment Approaches
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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)
ORCID: Liu, Xingrong
LeCluyse, Edward L.
Lemasters, John J.
Stieger, Bruno
Brouwer, Kim L. R.
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
DOI: 10.1152/ajpgi.1999.277.1.g12
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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Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration

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