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Drug Metabolism and Disposition Fast Forward
First published on June 16, 2006; DOI: 10.1124/dmd.105.009118

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Received for publication December 27, 2005.
Revised June 12, 2006.
Accepted for publication June 14, 2006.

Use of Cryopreserved Human Hepatocytes in Sandwich-Culture to Measure Hepatobiliary Transport

Yi-an Bi 1, Diana C Kazolias 1, David B Duignan 1*

1 Pfizer, Inc.

* Address correspondence to: E-mail: david.b.duignan{at}pfizer.com

Abstract

Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are: BioCoatTM plates, a cell density of 0.35 x 10-6 cells/well in 500 µL, an overlay of MatrigelTM and InVitroGROTM media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate (NTCP/OATPs uptake/BSEP efflux) the average apparent uptake (uptakeapp), apparent intrinsic biliary clearance (CLbile,int,app) and biliary excretion index (BEI) among five cryopreserved hepatocyte lots was high, ranging from 11-17 pmol/min/mg protein, 5.8 - 10 µL/min/mg protein and 41 - 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/MDR1 efflux) were 0.69 - 1.5 pmol/min/mg protein, 0.60 -1.5 µL/min/mg protein, and 37 - 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of BCRP and MRP2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.


Key words: drug transport, hepatic transport, hepatic uptake, hepatobiliary disposition, hepatobiliary transport, hepatocytes, transporters


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