Increased post-thaw viability and phase I and II biotransformation activity in cryopreserved rat liver slices after improvement of a fast-freezing method.
An existing cryopreservation method for liver slices applies 12% dimethylsulfoxide and rapid freezing. We found that cells in rat liver slices cryopreserved in this manner deteriorated rapidly upon culturing. To improve this cryopreservation method, we varied the dimethylsulfoxide concentration (0, 12, 18, and 30%), the cryopreservation medium (Williams medium E, fetal calf serum, and University of Wisconsin medium), slice thickness, and the storage period at 4 degrees C during slice preparation before cryopreservation. After thawing, slices were cultured for 4 h at 37 degrees C before their viability was evaluated by their potassium content and the number of intact cells determined histomorphologically. The biotransformation capacity of liver slices cryopreserved by the improved method was assessed by testosterone oxidation, hydroxycoumarin sulfation, and glucuronidation. Best results were obtained with 18% dimethylsulfoxide in Williams medium E: the potassium content of cryopreserved slices was higher than 65%, and the number of intact cells was higher than 60% of that in fresh slices; with 12% dimethylsulfoxide, potassium content was less than 40%, and the number of intact cells was less than 30%. Results did not differ between the three cryopreservation media. Viability of thin slices (8-10 cell layers) was better maintained than that of thicker slices (>14 cell layers). Storage at 4 degrees C of slices before cryopreservation decreased viability after cryopreservation. Both oxidative and conjugation activities were better than 60% of fresh values. Although results varied, slices cryopreserved with this improved method and cultured for 4 h retained viability between 50 and 80%, and biotransformation activity between 60 and 90% of fresh slices.
de Graaf
I A
IA
Solvay Pharmaceuticals BV, Drug Safety Department, The Netherlands. inge.degraaf@solvay.com
van der Voort
D
D
Brits
J H
JH
Koster
H J
HJ
D000818:Animals; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods / Q000592:standards*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005615:Freezing; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013739:Testosterone / Q000493:pharmacokinetics; D013997:Time Factors; D014021:Tissue Preservation
D013739:Testosterone; D011188:Potassium; D004121:Dimethyl Sulfoxide