Cryopreservation of adult rat and human hepatocytes for drug metabolism studies
Most of the information on the metabolism and toxicity of chemicals in man has been obtained by extrapolation from animal experiments. Hepatocytes isolated from laboratory animals have been used extensively for such experiments. The use of human hepatocytes for the prediction of liver xenobiotic metabolism and toxicity in man would avoid the problems encountered with species differences in drug metabolism during extrapolation of data from animals to man. However, the supply of suitable human liver material is both infrequent and unpredictable, and a means of prolonging the useful life-time of the isolated hepatocytes is necessary. The aim of this project was to use cryopreservation (preservation of biological material by freezing) and vitrification (low temperature preservation by solidification of liquid without freezing) to develop a method for the low temperature preservation of isolated rat and human adult hepatocytes, so that on thawing, they could be used for the study of xenobiotic metabolism and toxicity. 2) The first stage of development was to determine the optimum compositions of the preservation media to be used. A dimethyl- sulphoxide (DMSO) based cryoprotectant was studied. 3) Having optimised the compositon of the preservation media the effect of cooling rate on cell viability was determined. A cooling rate of 1°C/min to -80°C using -80°C subsequently as the storage temperature, was found to be the optimum freezing protocol using both cryoprotectants. 4) Using the VS25 and the DMSO cryoprotectant medium and the freezing schedule described above the viability of rat and human hepatocytes following cryopreservation periods of up to 1 month, was assessed by dye exclusion, reduced glutathione (GSH) and cytochrome P-450 content and the activities of a number of enzymes important in the activation and detoxification of xenobiotics. 5) Post-cryopreservation the hepatocytes will be used to study chemical toxicity in suspensions at 37°C and they should therefore be able to retain their viability under these conditions. 6) As a final assessment of their viability following cryopreservation, primary cultures of cryopreserved rat hepatocytes were initiated. After 24h culture on collagen poor attachment of the cryopreserved cells was obtained (17+3%), in comparison with freshly isolated hepatocytes (62+3%). 7)In conclusion, the low temperature preservation of isolated rat and, more importantly, human adult hepatocytes with high cellular viability on thawing was achieved. Although these cells can be used for short term metabolism/toxicity studies, caution must be exercised in interpreting data where glutathione transferase and reductase may be involved in the mechanism of toxicity. Two main problems remain to be solved. Firstly, the differential stability of the microsomal and cytosolic enzymes and secondly the loss of viability, particularly with rat cells, after incubation of cryopreserved cells at 37°C either in suspension or in primary culture.