Long-term maintenance of drug-metabolizing enzyme activities in rat hepatocytes after cryopreservation.
Recent studies have demonstrated that freshly isolated adult hepatocytes from various species can be hypothermically preserved for a short period or cryopreserved for a prolonged period before seeding in primary culture. This study was designed to determine whether rat hepatocytes could be maintained functional for a prolonged period after either hypothermic preservation or cryopreservation. Cold storage was carried out in University of Wisconsin solution (UW) and freezing in Leibovitz medium added with 10% fetal calf serum and 16% dimethyl sulfoxide. Rat hepatocytes were then set up either in pure conventional culture or in coculture with rat liver epithelial cells. Various functions were measured over 4- and 15-day periods, i.e., albumin secretion rate, deethylation of ethoxyresorufin and phenacetin, dealkylation of pentoxyresorufin, glucuronidation and sulfoconjugation of paracetamol, and N-acetylation of procainamide. No major differences were observed between unfrozen, frozen, and UW-preserved cells. While in pure culture all the functions tested were markedly decreased after 3 or 4 days, they remained high over the 15-day period in coculture, being either maintained or increased after 7-12 days compared to initial values. These results clearly demonstrate that when maintained under suitable culture conditions, rat hepatocytes can fully recover after hypothermic preservation or cryopreservation and therefore represent a suitable in vitro model system for pharmacotoxicological studies.
Fautrel
A
A
INSERM U 456, Université de Rennes I, Rennes, France.
Joly
B
B
Guyomard
C
C
Guillouzo
A
A
D000082:Acetaminophen / Q000737:chemistry / Q000378:metabolism; D000107:Acetylation; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000032:analysis / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology; D005965:Glucuronates / Q000737:chemistry; D008099:Liver / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D010615:Phenacetin / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D005965:Glucuronates; D000082:Acetaminophen; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D010615:Phenacetin; D004121:Dimethyl Sulfoxide