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

Shibany, Khaled A.; Tötemeyer, Sabine; Pratt, Stefanie L.; Paine, Stuart W.

Authors

Khaled A. Shibany

Stefanie L. Pratt



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 http://eprints.nottingham.ac.uk/id/eprint/37532
Publisher URL http://onlinelibrary.wiley.com/doi/10.1002/prp2.268/abstract
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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