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Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. KU Leuven Dept Pharmaceut & Pharmacol Sci, Drug Delivery & Disposit, Gasthuisberg O&N2 Herestr 49,Box 921, B-3000 Leuven, Belgium.
KU Leuven Dept Pharmaceut & Pharmacol Sci, Drug Delivery & Disposit, Gasthuisberg O&N2 Herestr 49,Box 921, B-3000 Leuven, Belgium.
KU Leuven Dept Pharmaceut & Pharmacol Sci, Drug Delivery & Disposit, Gasthuisberg O&N2 Herestr 49,Box 921, B-3000 Leuven, Belgium;Genentech Inc, San Francisco, CA 94080 USA.
KU Leuven Dept Pharmaceut & Pharmacol Sci, Drug Delivery & Disposit, Gasthuisberg O&N2 Herestr 49,Box 921, B-3000 Leuven, Belgium.ORCID iD: 0000-0003-2595-388X
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2018 (English)In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 20, no 2, article id 33Article in journal (Refereed) Published
Abstract [en]

Freshly-isolated rat hepatocytes are commonly used as tools for hepatic drug disposition. From an ethical point of view, it is important to maximize the use of isolated hepatocytes by cryopreservation. The present study compared overall hepatocyte functionality as well as activity of the organic anion transporting polypeptide (Oatp), multidrug resistance-associated protein 2 (Mrp2), and UDP-glucuronosyltransferase 1 (Ugt1), in in vitro models established with cryopreserved and freshly-isolated hepatocytes. A similar culture time-dependent decline in cellular functionality, as assessed by urea production, was observed in sandwich-cultured hepatocytes (SCH) obtained from freshly-isolated and cryopreserved cells. Concentration-dependent uptake kinetics of the Oatp substrate sodium fluorescein in suspended hepatocytes (SH) or SCH were not significantly affected by cryopreservation. Mrp2-mediated biliary excretion of 5 (and 6)-carboxy-2', 7'-dichlorofluorescein by SCH was assessed with semi-quantitative fluorescence imaging: biliary excretion index values increased between day 3 and day 4, but did not differ significantly between cryopreserved and freshly-isolated hepatocytes. Finally, telmisartan disposition was evaluated in SCH to simultaneously explore Oatp, Ugt1, and Mrp2 activity. In order to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation, a mechanistic cellular disposition model was developed. Basolateral and canalicular efflux as well as glucuronidation of telmisartan were affected by cryopreservation. In contrast, the disposition parameters of telmisartan-glucuronide were not impacted by cryopreservation. Overall, the relative contribution of the rate-determining processes (uptake, metabolism, efflux) remained unaltered between cryopreserved and freshly-isolated hepatocytes, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes when studying these cellular disposition pathways.

Place, publisher, year, edition, pages
2018. Vol. 20, no 2, article id 33
Keywords [en]
cryopreservation, Mrp2, Oatp, rat hepatocytes, sandwich-culture, suspended hepatocytes, telmisartan, UGT
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-387472DOI: 10.1208/s12248-018-0188-7ISI: 000427539900014PubMedID: 29468289OAI: oai:DiVA.org:uu-387472DiVA, id: diva2:1329415
Available from: 2019-06-24 Created: 2019-06-24 Last updated: 2019-06-24Bibliographically approved

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Keemink, Janneke

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