Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies

Shibany, Khaled A.; T�temeyer, Sabine; Pratt, Stefanie L.; Paine, Stuart W.

Abstract

Despite reports of the successful isolation of primary equine hepatocytes, thereare no published data regarding the successful cryopreservation of these isolatedcells. In this study, a detailed description of the procedures for isolation, cryop-reservation, and recovery of equine hepatocytes are presented. Furthermore, theintrinsic clearance (Clint) and production of metabolites for three drugs werecompared between freshly isolated and recovered cryopreserved hepatocytes. Pri-mary equine hepatocytes were isolated using a two-step collagenase perfusionmethod, with an average cell yield of 2.47  2.62 9 106cells/g of perfused livertissue and viability of 84.1  2.62%. These cells were cryopreserved with Wil-liam’s medium E containing 10% fetal bovine serum with 10% DMSO. The via-bility of recovered cells, after a 30% Percoll gradient, was 77  11% andestimated recovery rate was approximately 27%. These purified cells were usedto determine the in vitro Clintof three drugs used in equine medicine; omepra-zole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopre-served suspensions gave a comparable estimation of Clintcompared to fresh cellsfor these three drugs as well as producing the same metabolites. This work pavesthe way for establishing a bank of cryopreserved equine hepatocytes that can beused for estimating pharmacokinetic parameters such as the hepatic metabolicin vivo clearance of a drug as well as producing horse-specific drug metabolites.

Citation

Shibany, K. A., Tötemeyer, S., Pratt, S. L., & Paine, S. W. (2016). Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies. Pharmacology Research and Perspectives, 4(5), 1-8. https://doi.org/10.1002/prp2.268

• Journal Article Type: Article
• Acceptance Date: Aug 27, 2016
• Online Publication Date: Sep 30, 2016
• Publication Date: Sep 30, 2016
• Deposit Date: Oct 17, 2016
• Publicly Available Date: Oct 17, 2016
• Journal: Pharmacology Research and Perspectives
• Electronic ISSN: 2052-1707
• Publisher: Wiley Open Access
• Peer Reviewed: Peer Reviewed
• Volume: 4
• Issue: 5
• Article Number: e00268
• Pages: 1-8
• DOI: https://doi.org/10.1002/prp2.268
• Keywords: Cell viability; cryopreservation; drug metabolism; equine; hepatocyte isolation
• Public URL: https://nottingham-repository.worktribe.com/output/808673
• Publisher URL: http://onlinelibrary.wiley.com/doi/10.1002/prp2.268/abstract
• Contract Date: Oct 17, 2016