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Research ArticleArticle

Retention of Transporter Activities in Cryopreserved, Isolated Rat Hepatocytes

Robert Houle, Jennifer Raoul, Jean-François Lévesque, K. Sandy Pang, Deborah A. Nicoll-Griffith and Jose M. Silva
Drug Metabolism and Disposition April 2003, 31 (4) 447-451; DOI: https://doi.org/10.1124/dmd.31.4.447
Robert Houle
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Jennifer Raoul
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Jean-François Lévesque
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K. Sandy Pang
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Deborah A. Nicoll-Griffith
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Jose M. Silva
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Abstract

The success of cryopreservation of isolated hepatocytes with existing methodologies is assessed with respect to the retentivity of cell integrity/viability (defined by trypan blue) and metabolic activities upon thawing in comparison to those of freshly prepared cells. But the ability of the cryopreserved cells to transport xenobiotics relative to that of freshly prepared cells has not been investigated. In this study, we optimized our previous methodology for cryopreservation and evaluated the metabolism and transport of thawed hepatocytes. Half of the freshly, isolated rat hepatocytes prepared by collagenase perfusion were immediately used for studies of transport of [14C]taurocholate, [3H]estrone sulfate and [3H]estradiol 17β-d-glucuronide (1 μM) and metabolism of 7-hydroxy-4-(trifluoromethyl)-coumarin (100 μM), (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (250 μM), bufuralol (100 μM), and tolbutamide (100 μM), probes for UDP-glucuronyl transferase (UGT) and CYP3A, CYP2D, and CYP2C, respectively. The remaining half was cryopreserved using an optimized, programmed-freezing protocol, which was developed to minimize the prolonged release of latent heat during freezing. With the exception of the UGT probe, no significant difference (P > 0.05) was found in both metabolism and transport with freshly isolated versus cryopreserved hepatocytes upon thawing. In conclusion, we have demonstrated for the first time that thawed rat hepatocytes cryopreserved by a programmed-freezing protocol retain drug transport activities.

Footnotes

  • ↵1 Present Address: Merck Frosst Centre for Therapeutic Research and Co., P.O BOX 1005 Pointe-Claire-Dorval, QC, Canada, H9R 4P8.

  • Abbreviations used are::
    NTCP
    sodium-dependent taurocholate cotransporting polypeptide
    OATP
    organic anion-transporting polypeptide
    MRP2
    multi-drug resistance-associated transporter
    DMSO
    dimethyl sulfoxide
    BSA
    bovine serum albumin
    7-HFC
    7-hydroxy-4-(trifluoromethyl)-coumarin
    DFB
    (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one
    UGT
    UDP-glucuronyl transferase
    • Received September 3, 2002.
    • Accepted January 2, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 31 (4)
Drug Metabolism and Disposition
Vol. 31, Issue 4
1 Apr 2003
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Research ArticleArticle

Retention of Transporter Activities in Cryopreserved, Isolated Rat Hepatocytes

Robert Houle, Jennifer Raoul, Jean-François Lévesque, K. Sandy Pang, Deborah A. Nicoll-Griffith and Jose M. Silva
Drug Metabolism and Disposition April 1, 2003, 31 (4) 447-451; DOI: https://doi.org/10.1124/dmd.31.4.447

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Research ArticleArticle

Retention of Transporter Activities in Cryopreserved, Isolated Rat Hepatocytes

Robert Houle, Jennifer Raoul, Jean-François Lévesque, K. Sandy Pang, Deborah A. Nicoll-Griffith and Jose M. Silva
Drug Metabolism and Disposition April 1, 2003, 31 (4) 447-451; DOI: https://doi.org/10.1124/dmd.31.4.447
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