Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation.
Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were purified by a subsequent Percoll centrifugation. The isolated PC had a viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were cryopreserved with an optimized protocol in a computer-controlled freezer. After thawing, the PC still retained a viability of 89%. The activities of representative xenobiotic metabolizing enzymes were compared between freshly isolated and cryopreserved PC after thawing. The cytochrome P450 content and the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of testosterone in intact cells, were not affected by the cryopreservation. The following phase II enzyme activities were also well maintained after cryopreservation: Phenol sulfotransferase (92%), 1-naphthol UDP-glucuronosyl transferase (95%), soluble epoxide hydrolase (87%), and glutathione S-transferase (88%), determined with broad spectrum substrate 1-chloro-2,4-dinitrobenzene. However, there was a significant decrease in plating efficiency between freshly isolated (86%) and cryopreserved (57%) PC when they were cultured. The initial quality of the freshly isolated PC is decisive for the success of cryopreservation. These results support the use of cryopreserved PC in pharmacology and toxicology with the aim to reduce the number of experimental animals used.
Institute of Toxicology, University of Mainz, Germany.
B
B
Diener
H
H
Abdel-Latif
M
M
Arand
F
F
Oesch
D000826:Animal Testing Alternatives; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015239:Arylsulfotransferase / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003198:Computer Simulation; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003580:Cytochromes; D004492:Edetic Acid / Q000737:chemistry; D004851:Epoxide Hydrolases / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006900:Hydroxylation; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism
D003580:Cytochromes; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D004492:Edetic Acid; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase; D015239:Arylsulfotransferase; D004851:Epoxide Hydrolases; D014343:Trypan Blue