Abstract
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°C during slice preparation before cryopreservation. After thawing, slices were cultured for 4 h at 37°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°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.
Footnotes
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Send reprint requests to: I. A. M. de Graaf, Drug Safety Department, Solvay Pharmaceuticals BV, P.O. Box 900, 1380 DA Weesp, The Netherlands. E-mail: inge.degraaf{at}solvay.com
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This study was supported by a grant from the Dutch Platform for Alternatives for Animal experimentation.
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↵2 Theoretically, it is expected that freezing and thawing of slices determine viability after cryopreservation and not the storage period in liquid nitrogen (Mazur, 1984). We compared viability of slices stored for 30 min with slices from the same liver that were stored for approximately 48 h. Indeed, no differences were found.
- Abbreviations used are::
- DMSO
- dimethylsulfoxide
- IIF
- intracellular ice-crystal formation
- UW
- University of Wisconsin medium
- WME
- Williams medium E
- FCS
- fetal calf serum
- Received February 2, 2000.
- Accepted May 11, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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