title: Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.
abstract: Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.
authors.affiliation: Bioreactor Group, Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin, Berlin, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Lübberstedt
authors.affiliation: Pharmacelsus GmbH, Saarbrücken, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Müller-Vieira
authors.affiliation: Pharmacelsus GmbH, Saarbrücken, Germany.
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Biemel
authors.affiliation: DMPK Innovative Medicines, AstraZeneca R&D, Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Darnell
authors.affiliation: Bioreactor Group, Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin, Berlin, Germany.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Hoffmann
authors.affiliation: Bioreactor Group, Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin, Berlin, Germany.
authors.forename: Fanny
authors.initials: F
authors.lastname: Knöspel
authors.affiliation: Bioreactor Group, Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin, Berlin, Germany.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Wönne
authors.affiliation: Cytonet GmbH & Co KG, Weinheim, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Knobeloch
authors.affiliation: Department of Traumatology, Eberhard Karls University, Tübingen, Germany.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Nüssler
authors.affiliation: Departments of Surgery and of Bioengineering, McGowan Institute for Regenerative Medicine, University of Pittsburgh, USA.
authors.forename: Jörg C
authors.initials: JC
authors.lastname: Gerlach
authors.affiliation: DMPK Innovative Medicines, AstraZeneca R&D, Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Tommy B
authors.initials: TB
authors.lastname: Andersson
authors.affiliation: Bioreactor Group, Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin, Berlin, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Zeilinger
mesh_terms: D019149:Bioreactors; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008567:Membranes, Artificial; D008904:Miniaturization
chemical_list: D016895:Culture Media, Serum-Free; D008567:Membranes, Artificial; D003577:Cytochrome P-450 Enzyme System