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Enzyme induction in cryopreserved human hepatocyte cultures
Sabine Kafert-Kasting1, Krassimira Alexandrova, Marc Barthold
1Cytonet GmbH & Co., KG Hannover Branch, Feodor Lynen Strasse 21, D-30625 Hannover, Germany. sabine.kafert-kasting@cytonet.de
Toxicology
|February 14, 2006
Summary
Cryopreserved human hepatocytes cultured on collagen gels maintain similar cytochrome P450 enzyme induction responses to rifampicin as fresh cells. This method offers a viable alternative for drug candidate testing despite reduced basal enzyme activity.
Area of Science:
- Hepatocyte biology
- Drug metabolism
- Cryopreservation techniques
Background:
- Freshly isolated human hepatocytes are the gold standard for in vitro drug testing.
- Cryopreserved hepatocyte suspensions have limitations in cell attachment and viability.
- A novel method for cryopreserving hepatocytes on a collagen gel was developed.
Purpose of the Study:
- To establish and evaluate a technique for cryopreserving human hepatocytes on a collagen gel.
- To assess the morphology, basal enzyme activity, and inducible enzyme activity of cryopreserved hepatocytes.
- To compare the performance of cryopreserved hepatocytes with non-frozen cells in drug metabolism assays.
Main Methods:
- Human hepatocytes were cultured on a collagen gel and then cryopreserved.
- Post-thaw, damaged cells were removed, and an upper gel layer was added.
- Morphology was assessed, and basal and rifampicin-induced cytochrome P450 isoform activities (testosterone hydroxylation) were measured.
Main Results:
- Hepatocyte morphology after cryopreservation and thawing was comparable to non-frozen cells.
- Basal activities of cytochrome P450 isoforms were reduced in cryopreserved hepatocytes compared to non-frozen cells.
- Enzyme induction factors for CYP3A4 by rifampicin were similar between cryopreserved and non-frozen hepatocytes.
Conclusions:
- Human hepatocytes cryopreserved on collagen gels retain significant CYP3A4 inducibility by rifampicin.
- This cryopreservation method provides a stable in vitro model for drug metabolism studies.
- While basal activities are reduced, the preserved inductive capacity makes these cells valuable for drug candidate evaluation.
