Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells.
Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 10(6) KC, 2.7 × 10(5) LEC and 4.7 × 10(5) HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4-5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor.
Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.
Elisa
E
Pfeiffer
Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.
Victoria
V
Kegel
Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.
Katrin
K
Zeilinger
IfADo - Leibniz Research Centre for Working Environment and Human Factors at Dortmund Technical University, 44139 Dortmund, Germany.
Jan G
JG
Hengstler
Eberhard-Karls University Tübingen, BG Trauma Center, 72076 Tübingen, Germany.
Andreas K
AK
Nüssler
Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.
Daniel
D
Seehofer
Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany georg.damm@charite.de.
Georg
G
Damm
D002448:Cell Adhesion; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D008099:Liver / Q000166:cytology*; D008855:Microscopy, Electron, Scanning; D010587:Phagocytosis
Liver; liver in vitro model; liver tissue engineering; non-parenchymal liver cells; primary cell isolation; primary human hepatocytes