Hypothermic preservation of hepatocytes. III. Effects of resuspension media on viability after up to 7 days of storage.
Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. Using these studies, improved methods of hypothermic liver preservation for transplantation may be developed. In this study, rat hepatocytes were cold-stored for up to 7 days in University of Wisconsin liver preservation solution. At the end of each day of storage hepatocytes were resuspended in Krebs-Henseleit buffer or tissue-culture medium (Liebovitz-15; Fischer's; modified Fischer's, which was similar to Fischer's but with glycine and cysteine added; or Waymouth's medium). Hepatocyte viability was assessed by rewarming and oxygenating the suspensions and measuring the percentage of leakage of lactate dehydrogenase from the cells, the cellular concentration of potassium and the stimulation of respiration by succinate, all measures of plasma membrane integrity. Additionally, concentrations of ATP and glutathione after rewarming and reoxygenation in the various resuspension media were measured. Hepatocyte permeability to lactate dehydrogenase did not increase during cold storage of 1 to 7 days (7.2% +/- 2% leakage), indicating that most of the hepatocytes remained viable during cold storage. However, when rewarmed, loss of viability (leakage of lactate dehydrogenase) was dependent on the composition of the resuspension media. In Krebs-Henseleit buffer, viability was reduced after 2 and 3 days of storage (lactate dehydrogenase leakage on rewarming = 70% to 90%). Leakage of lactate dehydrogenase was reduced significantly after resuspension in tissue-culture media. After 6 days of storage, lactate dehydrogenase leakage from hepatocytes stored in Liebovitz- 15 or modified Fischer's was only about 30%.(ABSTRACT TRUNCATED AT 250 WORDS)
Marsh
D C
DC
Department of Surgery, University of Wisconsin, Madison 53792.
Hjelmhaug
J A
JA
Vreugdenhil
P K
PK
Kerr
J A
JA
Rice
M J
MJ
Belzer
F O
FO
Southard
J H
JH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002470:Cell Survival; D005978:Glutathione / Q000378:metabolism; D007036:Hypothermia, Induced; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D009926:Organ Preservation; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012119:Respiration; D013997:Time Factors
D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D011188:Potassium