Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies.
Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver.
Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany.
Stefan A
SA
Hoffmann
Ursula
U
Müller-Vieira
Klaus
K
Biemel
Daniel
D
Knobeloch
Sandra
S
Heydel
Marc
M
Lübberstedt
Andreas K
AK
Nüssler
Tommy B
TB
Andersson
Jörg C
JC
Gerlach
Katrin
K
Zeilinger
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000493:pharmacokinetics; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004008:Diclofenac / Q000493:pharmacokinetics; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008904:Miniaturization / Q000295:instrumentation*; D010599:Pharmacokinetics; D010600:Pharmacology / Q000295:instrumentation / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System