Metabolic activity of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes.
1. The effect of cryopreservation on the metabolic capacity of monkey hepatocytes over 4 h in suspension and 24 h in culture was determined. Hepatocytes were diluted in a buffer containing 10% DMSO and frozen in a computer-controlled chamber. 2. Initial ethoxyresorufin and ethoxycoumarin O-deethylase (ECOD) activities were the same in fresh and cryopreserved (CP) hepatocytes. ECOD activity in suspensions declined over 4 h but was the same in fresh and CP hepatocytes. 3. The formation of testosterone hydroxy (OHT) metabolites (namely 6beta-OHT, 2beta-OHT, 16beta-OHT, 16alpha-OHT, 15beta-OHT, 2alpha-OHT and 6beta-OHT) was unaffected by cryopreservation. The loss of OHT activities over 4 h in CP and fresh whole cell suspensions was attributed to a loss of cofactor. CP hepatocyte cultures had equivalent OHT activities to freshly isolated hepatocytes. 4. Initial UDP-glucuronyltransferase (UGT) activities, using the substrates 4-methylumbelliferone, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP whole hepatocytes. At later times, UGT activity was lower in CP than fresh hepatocytes but this was due to a loss of UDPGA. Initial sulphotransferase (SULT) activities, using the substrates 2-naphthol, ethoxycoumarin and hydroxycoumarin, were equivalent in fresh and CP hepatocytes. SULT activities were less stable than UGT activities but were the same in fresh and CP hepatocytes throughout the 4-h incubation. 5. Initial glutathione S-transferase activities (using 1-chloro-2,4-dinitrobenzene) were the same in fresh and CP hepatocytes and both did not decrease over 4 h. 6. CP monkey hepatocytes are a useful model for metabolic and cytotoxicity studies. These cells can be can be used either in suspension or in culture.
Hewitt
N J
NJ
Institute of Toxicology, Merck KGaA, Darmstadt, Germany. nicola.hewitt@merck.de
Fischer
T
T
Zuehlke
U
U
Oesch
F
F
Utesch
D
D
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002448:Cell Adhesion; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008252:Macaca fascicularis; D008297:Male; D013250:Steroid Hydroxylases / Q000378:metabolism
D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C022724:testosterone 7-alpha-hydroxylase, hamster; D005978:Glutathione