Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition
Abstract
Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Culturing hepatocytes in a sandwich configuration between two layers of extracellular matrix allows them to regain polarity and permits functional analysis of hepatocyte biliary excretion.
“Culturing hepatocytes in a sandwich configuration between two layers of extracellular matrix (ECM) allows them to regain polarity and permits functional analysis of hepatocyte biliary excretion (Deharde et al., 2016[8]; Fardel et al., 2019[11]).”
From Optimization of extracellular matrix for primary human hepatocyte cultures using mixed collagen-matrigel matrices (2023) · cites this paper as Deharde et al., 2016[8]
A previous study observed that application of Matrigel in combination with classic collagen can have a beneficial influence on hepatocyte morphology and polarization.
“In a previous study, we observed that the application of Matrigel in combination with the classic collagen culture can have a beneficial influence on hepatocyte morphology and polarization (Deharde et al., 2016[8]).”
From Optimization of extracellular matrix for primary human hepatocyte cultures using mixed collagen-matrigel matrices (2023) · cites this paper as Deharde et al., 2016[8]
In accordance with former observations, the tight junction and apical domain markers ZO-1 and MRP2 were expressed diffusely in the pure Matrigel SCs.
“In accordance with our former observations (Deharde et al., 2016[8]), the tight junction and apical domain markers ZO-1 and MRP2 were expressed diffusely in the pure Matrigel SCs.”
From Optimization of extracellular matrix for primary human hepatocyte cultures using mixed collagen-matrigel matrices (2023) · cites this paper as Deharde et al., 2016[8]
Cites
Godoy, P., Hewitt, N. J., Albrecht, U., Andersen, M. E., Ansari, N., Bhattacharya, S., Bode, J. G., Bolleyn, J., Borner, C., Böttger, J., et al. (2013) · Archives of Toxicology · doi:10.1007/s00204-013-1078-5 · Cited by 1,280 · see citing works
Self-citationVukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B., Roche, N. S., & Reddi, A. H. (1992) · Experimental Cell Research · doi:10.1016/0014-4827(92)90397-q · Cited by 696 · see citing works
Long‐Term in Vitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration
Dunn, J., Tompkins, R. G., & Yarmush, M. L. (1991) · Biotechnology Progress · doi:10.1021/bp00009a007 · Cited by 687 · see citing works
Rajan, N., Habermehl, J., Côté, M., Doillon, C. J., & Mantovani, D. (2006) · Nature Protocols · doi:10.1038/nprot.2006.430 · Cited by 579 · see citing works
LeCluyse, E. L. (2001) · European Journal of Pharmaceutical Sciences · doi:10.1016/s0928-0987(01)00135-x · Cited by 477 · see citing works
Francois Berthiaume, B., Moghe, P. V., Toner, M., & Yarmush, M. L. (1996) · The FASEB Journal · doi:10.1096/fasebj.10.13.8940293 · Cited by 397 · see citing works
Swift, B., Pfeifer, N. D., & Brouwer, K. L. R. (2010) · Drug Metabolism Reviews · doi:10.3109/03602530903491881 · Cited by 374 · see citing works
Elaut, G., Henkens, T., Papeleu, P., Snykers, S., Vinken, M., Vanhaecke, T., & Rogiers, V. (2006) · Current Drug Metabolism · doi:10.2174/138920006778017759 · Cited by 292 · see citing works
Treyer, A., & Müsch, A. (2013) · Comprehensive Physiology · doi:10.1002/cphy.c120009 · Cited by 252 · see citing works
The hepatic extracellular matrix
Martínez‐Hernández, A., & Amenta, P. S. (1993) · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin · doi:10.1007/bf01606580 · Cited by 238 · see citing works
Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function
Moghe, P. V., Francois Berthiaume, B., Ezzell, R. M., Toner, M., Tompkins, R. G., & Yarmush, M. L. (1996) · Biomaterials · doi:10.1016/0142-9612(96)85576-1 · Cited by 227 · see citing works
Use of Cryopreserved Human Hepatocytes in Sandwich Culture to Measure Hepatobiliary Transport
Bi, Y.-A., Kazolias, D., & Duignan, D. B. (2006) · Drug Metabolism and Disposition · doi:10.1124/dmd.105.009118 · Cited by 216 · see citing works
The hepatic extracellular matrix: I. Components and distribution in normal liver
Martinez-Hernandez, A., & Amenta, P. S. (1993) · Virchows Archiv A Pathological Anatomy and Histopathology · doi:10.1007/bf01606425 · Cited by 200 · see citing works
Coon, J., Hamilton, G. A., Jolley, S. L., Gilbert, D., Barros, S. A., & LeCluyse, E. L. (2001) · Cell and Tissue Research · doi:10.1007/s004410100429 · Cited by 192 · see citing works
Toxicogenomics directory of chemically exposed human hepatocytes
Grinberg, M., Stöber, R. M., Edlund, K., Rempel, E., Godoy, P., Reif, R., Widera, A., Madjar, K., Schmidt‐Heck, W., Marchan, R., et al. (2014) · Archives of Toxicology · doi:10.1007/s00204-014-1400-x · Cited by 139 · see citing works
Self-citationUse of Ca2+ Modulation to Evaluate Biliary Excretion in Sandwich-Cultured Rat Hepatocytes
Liu, X., LeCluyse, E. L., Brouwer, K. R., Lightfoot, R. M., Lee, J. I., & Brouwer, K. L. R. (1999) · Journal of Pharmacology and Experimental Therapeutics · doi:10.1016/s0022-3565(24)38310-7 · Cited by 133 · see citing works
Schyschka, L., Sánchez, J. M., Wang, Z., Burkhardt, B., Müller‐Vieira, U., Zeilinger, K., Bachmann, A., Nadalin, S., Damm, G., & Nussler, A. K. (2013) · Archives of Toxicology · doi:10.1007/s00204-013-1080-y · Cited by 128 · see citing works
Self-citationRegulation of the blood?biliary barrier: interaction between gap and tight junctions in hepatocytes
Kojima, T., Yamamoto, T., Murata, M., Chiba, H., Kokai, Y., & Sawada, N. (2003) · Medical Molecular Morphology · doi:10.1007/s00795-003-0220-5 · Cited by 125 · see citing works
Bruyn, T. D., Chatterjee, S., Fattah, S., Keemink, J., Nicolaï, J., Augustijns, P., & Annaert, P. (2013) · Expert Opinion on Drug Metabolism & Toxicology · doi:10.1517/17425255.2013.773973 · Cited by 124 · see citing works
Pfeiffer, E., Kegel, V., Zeilinger, K., Hengstler, J. G., Nussler, A. K., Seehofer, D., & Damm, G. (2014) · Experimental Biology and Medicine · doi:10.1177/1535370214558025 · Cited by 104 · see citing works
Self-citationEpithelial polarity – Generating and integrating signals from the ECM with integrins
Manninen, A. (2015) · Experimental Cell Research · doi:10.1016/j.yexcr.2015.01.003 · Cited by 103 · see citing works
Page, J. L., Johnson, M. C., Olsavsky, K. M., Strom, S. C., Zarbl, H., & Omiecinski, C. J. (2007) · Toxicological Sciences · doi:10.1093/toxsci/kfm034 · Cited by 77 · see citing works
Abe, K., Bridges, A. S., & Brouwer, K. L. R. (2008) · Drug Metabolism and Disposition · doi:10.1124/dmd.108.023465 · Cited by 74 · see citing works
Hoffmann, S. A., Müller‐Vieira, U., Biemel, K. M., Knobeloch, D., Heydel, S., Lübberstedt, M., Nussler, A. K., Andersson, T. B., Gerlach, J. C., & Zeilinger, K. (2012) · Biotechnology and Bioengineering · doi:10.1002/bit.24573 · Cited by 69 · see citing works
Self-citationThe unique polarity phenotype of hepatocytes
Müsch, A. (2014) · Experimental Cell Research · doi:10.1016/j.yexcr.2014.06.006 · Cited by 64 · see citing works
血液‐胆管バリアの調節:肝細胞でのギャップ結合と密着結合の相互作用
Kojima, T., Yamamoto, T., Murata, M., Chiba, H., Kokai, Y., & Sawada, N. (2003) · Cited by 64 · see citing works
Human Hepatocytes: Isolation, Culture, and Quality Procedures
Knobeloch, D., Ehnert, S., Schyschka, L., Büchler, P., Schoenberg, M., Kleeff, J. H., Thasler, W. E., Nüssler, N. C., Godoy, P., Hengstler, J. G., et al. (2011) · Methods in molecular biology · doi:10.1007/978-1-61779-367-7_8 · Cited by 63 · see citing works
Self-citationBi, Y., Kimoto, E., Sevidal, S., et al. (2012) · Drug Metabolism and Disposition · doi:10.1124/dmd.111.043489 · Cited by 60 · see citing works
Turncliff, 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
Bile 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
Self-citationHoffmaster, 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
Swift, B., & Brouwer, K. L. R. (2009) · Molecular Pharmaceutics · doi:10.1021/mp900227a · Cited by 45 · see citing works
Groß‐Steinmeyer, K., Stapleton, P. L., Tracy, J. H., Bammler, T. K., Lehman, T., Strom, S. C., & Eaton, D. L. (2005) · Xenobiotica · doi:10.1080/00498250500137427 · Cited by 42 · see citing works
Improved Hepatocyte Excretory Function by Immediate Presentation of Polarity Cues
Ng, S., Han, R., Chang, S., et al. (2006) · Tissue Engineering · doi:10.1089/ten.2006.12.2181 · Cited by 32 · see citing works
Boyer, J. L. (1997) · Cell Biology and Toxicology · doi:10.1023/a:1007435408208 · Cited by 19 · see citing works
Gillette, K., Forbes, K., & Sehgal, I. (2003) · Tumori Journal · doi:10.1177/030089160308900415 · Cited by 13 · see citing works
Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells
Pfeiffer, E., Kegel, V., Zeilinger, K., Hengstler, J. G., Nussler, A. K., Seehofer, D., & Damm, G. (2014) · Experimental Biology and Medicine · doi:10.15496/publikation-6936 · Cited by 10 · see citing works
Self-citation
Flagged first, then by citations
Cited by
Sorted by citations
Modeling alcohol-associated liver disease in a human Liver-Chip
Nawroth, J. C., Petropolis, D. B., Manatakis, D. V., Maulana, T. I., Burchett, G., Schlünder, K., Witt, A., Shukla, A., Kodella, K., Ronxhi, J., et al. (2021) · Cell Reports · doi:10.1016/j.celrep.2021.109393 · Cited by 88 · see citing works
Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements
Kaur, I., Vasudevan, A., Rawal, P., Tripathi, D. M., Ramakrishna, S., Kaur, S., & Sarin, S. K. (2023) · Bioengineering · doi:10.3390/bioengineering10020131 · Cited by 64 · see citing works
Seidemann, L., Prinz, S., Scherbel, J.-C., Götz, C., Seehofer, D., & Damm, G. (2023) · IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund · doi:10.17179/excli2022-5459 · Cited by 3 · see citing works
Self-citation
- Authors
- Deharde, D., Schneider, C., Hiller, T., Fischer, N., Kegel, V., Lübberstedt, M., Freyer, N., Hengstler, J. G., Andersson, T. B., Seehofer, D., Pratschke, J., Zeilinger, K., & Damm, G.
- Affiliations
- Stiftung Charité (DE)
- Berlin-Brandenburger Centrum für Regenerative Therapien (DE)
- Humboldt-Universität zu Berlin (DE)
- TU Dortmund University (DE)
- Karolinska Institutet (SE)
- Corresponding author
- Damm, Georg
- Venue
- Archives of Toxicology
- Published
- 2016-06-21
- Volume
- 90
- Issue
- 10
- Pages
- 2497-2511
- ISSN
- 0340-5761 (ISSN-L)
- 1432-0738
- OpenAlex
- W2468744958
- Semantic Scholar
- 4ecdb78b800373629fda1904302139be71c29819
- Cited by
- 58
- References
- 37
- Citations by year
- 2026: 1 · 2025: 6 · 2024: 4 · 2023: 4 · 2022: 8 · 2021: 8 · 2020: 6 · 2019: 4 · 2018: 11 · 2017: 5 · 2016: 1
- FWCI
- 4.25
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Archives of Toxicology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- BMBF Virtual Liver Network (0315741, 0315739)
- Topics
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Keywords
- biliary transport, primary human hepatocytes, sandwich culture, extracellular matrix composition, collagen, Matrigel, biliary excretion, hepatocyte morphology, cell-matrix interactions, cell-cell contact, hepatotoxicity testing
- MeSH terms
- Aged, Aged, 80 and over, Bile, Bile Canaliculi, Biological Transport, Cell Adhesion, Cell Survival, Cells, Cultured, Collagen, Drug Combinations, Extracellular Matrix, Female, Humans, Laminin, Male, Microscopy, Phase-Contrast, Middle Aged, Proteoglycans, Cell Polarity, Hepatocytes, Primary Cell Culture
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
