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% dimethylsu lfoxide and rapid freezing. We found that cells in rat liver slices cryopre served 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 cal f serum, and University of Wisconsin medium), slice thickness, and the stor age period at 4 degrees C during slice preparation before cryopreservation. After thawing, slices were cultured for 4 h at 37 degrees C before their v iability was evaluated by their potassium content and the number of intact cells determined histomorphologically. The biotransformation capacity of li ver slices cryopreserved by the improved method was assessed by testosteron e oxidation, hydroxycoumarin sulfation, and glucuronidation. Best results w ere obtained with 18% dimethylsulfoxide in Williams medium E: the potassium content of cryopreserved slices was higher than 65%, and the number of int act cells was higher than 60% of that in fresh slices; with 12% dimethylsul foxide, potassium content was less than 40%, and the number of intact cells was less than 30%. Results did not differ between the three cryopreservati on 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 s lices before cryopreservation decreased viability after cryopreservation. B oth oxidative and conjugation activities were better than 60% of fresh valu es. Although results varied, slices cryopreserved with this improved method and cultured for 4 h retained viability between 50 and 80%, and biotransfo rmation activity between 60 and 90% of fresh slices.