Cryopreservation of rat precision-cut liver slices is associated with major metabolic stress and ionic perturbations.
OBJECTIVE
Cryopreserved precision-cut liver slices (PCLS) in culture exhibit a rapid decline in ATP level and anabolic processes by unknown mechanisms. The aim of this study is to elucidate the key events explaining the alterations occurring in cryopreserved PCLS.


METHODS
Glucose metabolism, mitochondrial activities, ionic homeostasis and caspase-3 activity were assessed in fresh or cryopreserved (rapid freezing-thawing conditions) rat PCLS within the first hour of incubation in Williams' medium E at 37 degrees C.


RESULTS
Despite a similar glycolytic activity under both conditions, only fresh PCLS were able to gradually recover their ATP and potassium content. Glycogen content dropped more rapidly in cryopreserved than in control PCLS. Only cryopreserved PCLS exhibited a decline in O2 consumption and a lower ATP/ADP ratio from 30 min of incubation with a loss of coupling of oxygen consumption to ATP synthesis and mitochondrial calcium transport after 1 hour of incubation. Caspase-3 activation was already present in cryopreserved PCLS after a few minutes of incubation.


CONCLUSIONS
The lack of restoration of potassium/sodium exchange might be a primary event in the metabolic alterations occurring in cryopreserved PCLS after thawing, that contributes to further mitochondrial alterations, caspase activation and compromises ATP-dependent anabolic pathways.
Vanhulle
Valérie P
VP
Unit of Pharmacokinetics, Metabolism, Nutrition and Toxicology, Université Catholique de Louvain, Belgium.
Martiat
Geneviève A
GA
Bontemps
Françoise
F
Vincent
Marie-Françoise
MF
Pycke
Jean-Marie
JM
Verbeeck
Roger K
RK
Horsmans
Yves
Y
Delzenne
Nathalie
N
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D015925:Cryopreservation; D005615:Freezing; D005947:Glucose / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D050356:Lipid Metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D010101:Oxygen Consumption; D006180:Proton-Translocating ATPases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013312:Stress, Physiological / Q000378:metabolism*; D013995:Time
D007477:Ions; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D006180:Proton-Translocating ATPases; D005947:Glucose