Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies
Shibany, Khaled A. and Tötemeyer, Sabine and Pratt, Stefanie L. and Paine, Stuart W. (2016) Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies. Pharmacology Research and Perspectives, 4 (5). e00268. ISSN 2052-1707
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.
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