Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification.

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.

Gastric neuroendocrine tumors in a 2-year oncogenicity study with CD-1 mice.

Descriptions of two rare gastric neuroendocrine tumors (carcinoids) of enterochromaffin (ECL) cells in CD-1 mice (2/50) from a 104-week oncogenicity study of a serotonergic/dopaminergic compound are presented. These tumors were detected at necropsy and confirmed by histopathology in hematoxylin and eosin- and Chromogranin A-stained slides. ECL cell counts of the glandular stomachs were determined by quantitative image analysis and did not reveal any hyperplastic changes as possible predisposing lesions for carcinoid formation. To investigate the possibility of drug-induced hypergastrinemia as the cause of tumor formation of ECL cells, gastrin blood levels were measured after treating mice for 7 days with the test substance. In this study, Omeprazole, the positive control, raised gastrin levels, while the test material did not. It was concluded that these two tumors were an example of "late-life"-occurring, spontaneous neuroendocrine tumors in the stomachs of aged CD-1 mice.