title: Retention of transporter activities in cryopreserved, isolated rat hepatocytes.
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 [(14)C]taurocholate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide (1 microM) and metabolism of 7-hydroxy-4-(trifluoromethyl)-coumarin (100 microM), (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (250 microM), bufuralol (100 microM), and tolbutamide (100 microM), 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.
authors.affiliation: Merck Frosst Centre for Therapeutic Research and Co., PO Box 1005, Pointe-Claire-Dorval, Montréal, Québec H9R 4P8, Canada. robert_houle@merck.com
authors.forename: Robert
authors.initials: R
authors.lastname: Houle
authors.forename: Jennifer
authors.initials: J
authors.lastname: Raoul
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Lévesque
authors.forename: K Sandy
authors.initials: KS
authors.lastname: Pang
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Nicoll-Griffith
authors.forename: Jose M
authors.initials: JM
authors.lastname: Silva
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001692:Biological Transport; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D051544:Cytochrome P-450 CYP3A; D004121:Dimethyl Sulfoxide; D005069:Evaluation Studies as Topic; D022781:Hepatocytes / Q000378:metabolism*; D066298:In Vitro Techniques; D007554:Isotopes / Q000378:metabolism; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000378:metabolism; D013656:Taurocholic Acid / Q000378:metabolism; D013997:Time Factors; D014021:Tissue Preservation
chemical_list: D007554:Isotopes; D009004:Monosaccharide Transport Proteins; D013656:Taurocholic Acid; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D004121:Dimethyl Sulfoxide