Influence of 48 hours of cold storage in University of Wisconsin organ preservation solution on metabolic capacity of rat hepatocytes.
OBJECTIVE
Suspensions of isolated hepatocytes are a valuable tool to study liver functions. For optimal use of the isolated hepatocytes, methods are needed to preserve the hepatocytes while maintaining their viability, metabolic and transport functions. Until now, little has been known about the maintenance of the drug metabolism capacity and energy state, measured by the so-called energy charge (ATP+1/2ADP)/(ATP+ADP+AMP), in hepatocytes after storage in University of Wisconsin cold storage solution (UW). Consequently, we investigated whether UW, originally designed to preserve organs for transplantation, was suitable for preservation of isolated rat hepatocytes with respect to the maintenance of drug metabolism and levels of energy-rich substrates.


METHODS
Viability of the isolated rat hepatocytes was determined by trypan blue exclusion, ATP content and energy charge after 24 and 48 h of storage in UW at 0 degrees C. Phase I and II metabolic functions of the cells were studied by measuring the cytochrome P450 content and the metabolic rate of lidocaine and 7-ethoxycoumarin.


RESULTS
During 48 h of storage of hepatocytes in UW both phase I and phase II metabolism are preserved at control levels. After storage, the viability of the hepatocytes was not changed significantly, and the cells maintained proper cellular ATP content and overall energy charge.


CONCLUSIONS
These results imply that hepatocytes from a single isolation can be stored in UW solution and used for metabolism experiments for 3 consecutive days, allowing a reduction in the use of experimental animals.
Olinga
P
P
Groningen Institute for Drug Studies, Department of Pharmacokinetics and Drug Delivery, University Hospital, The Netherlands. peter@farm.rug.nl
Merema
M
M
Slooff
M J
MJ
Meijer
D K
DK
Groothuis
G M
GM
D000818:Animals; D003374:Coumarins / Q000494:pharmacology; D015925:Cryopreservation; D008012:Lidocaine / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D019188:Organ Preservation Solutions; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014495:Universities; D014922:Wisconsin
D003374:Coumarins; D019188:Organ Preservation Solutions; C017299:7-ethoxycoumarin; D008012:Lidocaine