Abstract
The research described in this thesis had two important aims. The first was to determine whether tissue slices could be used as an in vitro tool to predict the in vivo metabolism of new drugs. The second aim was to find a manner to store tissue slices for longer time
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periods by cryopreservation.
It was found that liver slices better than liver homogenate and microsomes predicted the in vivo metabolite pattern of drugs. One of the three drugs studied in this thesis, however, was found to be metabolized extra-hepatically and studying metabolism with liver slices alone gave an incomplete view of in vivo metabolism. The most important in vivo metabolite of the latter compound was found to be formed in great amounts by intestinal slices. We proposed a rough manner to combine the metabolite pattern produced by both liver and extra-hepatical slices to be able to use tissue slices in a prospective study for the prediction of in vivo drug metabolism. It was shown that in this manner, the in vivo metabolite pattern of the tested drugs was rightfully predicted.
For cryopreservation of tissue slices, both classical equilibrium freezing and vitrification methods were used. Both these methods tend to prevent the formation of potentially damaging intracellular ice. Besides, slices were frozen by rapid freezing. To test the viability of cryopreserved slices, several end-points were used. It was found that slice histomorphology, potassium and ATP content and phase II biotransformation were very sensitive to cryopreservation damage. Viability of liver slices, determined with these end-points, was successfully preserved by rapid freezing, despite of the fact that intra- and extracellular ice formation was not prevented. The liver slices did not survive equilibrium slow freezing. It was suggested that by rapid freezing and thawing small ice crystals are formed that can be resisted by the cells under certain conditions. This hypothesis was supported by the fact that when the rapidly frozen liver slices were warmed slowly, allowing small ice crystals to grow, the slices lost viability. With equilibrium slow freezing, intracellular ice crystal formation was probably prevented, but large ice crystals were allowed to form between the cells in the slices, damaging the cells from the outside.
Rapid freezing did not preserve viability of kidney and intestinal slices. Vitrification prevents the formation of damaging intra- and extracellular ice crystals by inducing the formation of a glass instead of ice. However, the toxicity of the highly concentrated CPAs required for vitrification was found to be a major concern. We showed that these problems could be overcome for a great part by using mixtures of CPAs like VM3 and VS4 and by adequately designing protocols for impregnation and out-washing of these compounds. Kidney and liver slices impregnated with VM3 and vitrified by slow cooling were found to survive cryopreservation. Therefore, a major conclusion of this thesis was that from a future perspective, vitrification as a starting point offers the highest chance to develop universal cryopreservation methods for tissue slices.
In conclusion, the results of the research described in this thesis indicate that organ slices can be vitrified to form a tissue slice bank from which they can be derived for in vitro drug metabolism studies, serving the reduction of laboratory animals use for drug safety studies and facilitating the use of human organ material.
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