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 as follows: BioCoat plates, a cell density of 0.35 × 106 cells/well in 500 μl, an overlay of Matrigel and InVitroGRO 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 [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 μl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 μl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.009118.
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ABBREVIATIONS: IVT, In Vitro Technologies, Inc.; HMM, hepatocyte maintenance media; WME, Williams' media E; DMEM, Dulbecco's modified Eagle's media; HBSS, Hanks' balanced salt solution; MRP (ABCC), human multidrug resistance-associated protein; MDR (ABCB), human multidrug resistance; BCRP, human breast cancer resistance protein; CDFDA, 5-(and-6)-carboxy-2′,7′-dichloro-fluorescein diacetate; EG, estradiol-17β-d-glucuronide; BSP, sulfobromo-phthalein; CDF, 5-(and-6)-carboxy-2′,7′-dichloro-fluorescein; FBS, fetal bovine serum; LC/MS/MS, liquid chromatography/tandem mass spectrometry; SN-38, 7-ethyl-10-hydroxycamptothecin; uptakeapp, apparent uptake rate; CLbile,int,app, apparent intrinsic biliary clearance; BEI, biliary excretion index; RC, rigid collagen; GC, gelled collagen; NTCP (SLC10A1), human Na+ taurocholate cotransporting polypeptide; OATP (SLCO), human organic anion transporting polypeptide; BSEP (ABCB11), human bile salt export pump.
- Received December 23, 2005.
- Accepted June 14, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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