Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.
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.
Department of General-, Visceral- and Transplantation Surgery, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Daniela
D
Deharde
Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Christin
C
Schneider
Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Thomas
T
Hiller
Mathematical and Science Faculty, Institute for Physics, Humboldt-Universität zu Berlin, Newtonstraße 15, 12489, Berlin, Germany.
Nicolas
N
Fischer
Department of General-, Visceral- and Transplantation Surgery, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Victoria
V
Kegel
Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Marc
M
Lübberstedt
Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Nora
N
Freyer
IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund Technical University, Ardeystraße 67, 44139, Dortmund, Germany.
Jan G
JG
Hengstler
DMPK, Cardiovascular and Metabolic Diseases Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, 43183, Mölndal, Sweden.|Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Solnavägen 1, 17177, Stockholm, Sweden.
Tommy B
TB
Andersson
Department of General-, Visceral- and Transplantation Surgery, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Daniel
D
Seehofer
Department of General-, Visceral- and Transplantation Surgery, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Johann
J
Pratschke
Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
Katrin
K
Zeilinger
Department of General-, Visceral- and Transplantation Surgery, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany. georg.damm@charite.de.
Georg
http://orcid.org/0000-0002-2104-8076
G
Damm
D000368:Aged; D000369:Aged, 80 and over; D001646:Bile / Q000378:metabolism*; D001648:Bile Canaliculi / Q000378:metabolism*; D001692:Biological Transport; D002448:Cell Adhesion; D016764:Cell Polarity; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000737:chemistry; D008297:Male; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D061251:Primary Cell Culture / Q000379:methods*; D011509:Proteoglycans / Q000737:chemistry
D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen
Biliary excretion; Extracellular matrix; Hepatocyte polarization; Primary human hepatocytes; Sandwich culture