Archives of Toxicology

, Volume 90, Issue 10, pp 2497–2511 | Cite as

Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition

  • Daniela Deharde
  • Christin Schneider
  • Thomas Hiller
  • Nicolas Fischer
  • Victoria Kegel
  • Marc Lübberstedt
  • Nora Freyer
  • Jan G. Hengstler
  • Tommy B. Andersson
  • Daniel Seehofer
  • Johann Pratschke
  • Katrin Zeilinger
  • Georg Damm
In vitro Systems

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.

Keywords

Primary human hepatocytes Sandwich culture Hepatocyte polarization Extracellular matrix Biliary excretion 

Notes

Acknowledgments

We would like to thank Melanie Kießig for her excellent technical assistance. This study was supported by the German Federal Ministry of Education and Research (BMBF) Project Virtual Liver: 0315741 and 0315739.

Supplementary material

204_2016_1758_MOESM1_ESM.tiff (455 kb)
Supplemental Fig. 1 Biliary excretion. Bile canaliculi from three different donors (A–C) cultured in four different combinations of collagen and Matrigel were treated with CDF-DA over 10 min, and CDF excretion was measured in the supernatant of the cultures over 48 min. Data show means of duplicates (n = 2) (TIFF 454 kb)
204_2016_1758_MOESM2_ESM.xlsx (42 kb)
Supplemental Table 1 Statistic evaluation of bile canaliculi formation. Filled bile canaliculi in sandwich-cultured hepatocytes after treatment with CDF-DA were quantified (Fig. 5) and statistically analyzed. Data show mean ± SD, p value and levels of significance of three independent experiments (N = 3; n = 3). (XLSX 42 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Daniela Deharde
    • 1
  • Christin Schneider
    • 2
  • Thomas Hiller
    • 2
  • Nicolas Fischer
    • 3
  • Victoria Kegel
    • 1
  • Marc Lübberstedt
    • 2
  • Nora Freyer
    • 2
  • Jan G. Hengstler
    • 4
  • Tommy B. Andersson
    • 5
    • 6
  • Daniel Seehofer
    • 1
  • Johann Pratschke
    • 1
  • Katrin Zeilinger
    • 2
  • Georg Damm
    • 1
  1. 1.Department of General-, Visceral- and Transplantation SurgeryCharité - Universitätsmedizin BerlinBerlinGermany
  2. 2.Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT)Charité - Universitätsmedizin BerlinBerlinGermany
  3. 3.Mathematical and Science Faculty, Institute for PhysicsHumboldt-Universität zu BerlinBerlinGermany
  4. 4.IfADo - Leibniz Research Centre for Working Environment and Human FactorsDortmund Technical UniversityDortmundGermany
  5. 5.DMPK, Cardiovascular and Metabolic Diseases Innovative MedicinesAstraZeneca R&DMölndalSweden
  6. 6.Section of Pharmacogenetics, Department of Physiology and PharmacologyKarolinska InstitutetStockholmSweden

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