Cryopreservation of rat precision-cut liver slices by ultrarapid freezing: influence on phase I and II metabolism and on cell viability upon incubation for 24 hours.
Several cryopreservation methods for precision-cut rat liver slices (PCLS) have been proposed, allowing a short-term (a few hours) maintainance of viability and functionality upon thawing. The aim of the present study was to test the metabolic capacity of PCLS cryopreserved by an ultrarapid method. The biotransformation of paracetamol to its glucuronide and sulfate conjugates and of midazolam to its hydroxylated metabolites was studied in thawed PCLS incubated for 24 hours at 37 degrees C in Williams' medium E. In addition, protein levels of the key enzymes involved in these metabolic reactions, i.e. UGT1A1, ST1A1, CYP2E1 and CYP3A2 were determinated. In addition, biological markers of cell function (ATP and glycogen levels) and toxicity (LDH leakage in the medium) were also measured. Compared to controls (non cryopreserved PCLS), CYP3A2 activity and content and CYP2E1 content were maintained at the same level all along the incubation, whereas paracetamol glucuronidation and sulfation dropped to 24 and 21% of the control value, respectively, immediately after thawing. Freezing-thawing conditions also modified cell functionality, leading to a lower intracellular ATP and glycogen content, and an increase in cell lysis, as shown by LDH released in the medium. The results of this study suggest that cryopreserved PCLS are able to maintain some phase I activities for 24 hours after thawing whereas some phase II metabolic capacities are not maintained.
Vanhulle
V P
VP
Unit of Pharmacokinetics, Metabolism, Nutrition and Toxicology, Université Catholique de Louvain, Brussels, Belgium.
Martiat
G A
GA
Verbeeck
R K
RK
Horsmans
Y
Y
Calderon
P B
PB
Eeckhoudt
S L
SL
Taper
H S
HS
Delzenne
N
N
D000082:Acetaminophen / Q000493:pharmacokinetics; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001711:Biotransformation; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005615:Freezing; D006003:Glycogen / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver; D008297:Male; D008874:Midazolam / Q000493:pharmacokinetics; D009924:Organ Culture Techniques; D009926:Organ Preservation / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar
D007527:Isoenzymes; D000082:Acetaminophen; D000255:Adenosine Triphosphate; D006003:Glycogen; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; D008874:Midazolam