TISSUE CULTURE RESEARCH COMMUNICATIONS

In pharmacology and toxicity evaluations for the first screening of a large number of new drugs, it becomes possible to deal, if detoxication of hepatocytes is used, even if the animal experiment is not carried out. In this study, electrochemical measurement of drug metabolism activity for the mass-screening system using cultured hepatocytes, in which low error by estimation was possible, was chosen and application as pharmacology and toxicity evaluation method was tried.
Mouse hepatocytes were cultured on an ITO electrode, the difference in aminopyrine as a substrate of CYP and |arge current change which depends for drug metabolism activity on the case in which aminoantipyrine which is the metabolite was added was able to be detected. On the other hand, the difference in remarkable current was not observed as a result of similar experiment using Hepa.1-6 of mouse hepatoma, in which drug metabolism activity extremely lowers, in comparison with hepatocytes. Therefore, current change observed in hepatocytes seems to be dependent on drug metabolism activity, and electrochemical measurement of drug metabo|iom activity of cultured hepatocytes, in which conveniently mass-screening in the short time was possible, could be established.

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The best condition for storage of cultured skin was investigate using two types of living skin models, i. e., (1) a three-dimensional culture skin (3DCS) consisting of both living human keratinocytes and fibroblasts and (2) a cultured dermis (CD) with only living fibroblasts. The biological activity of each skin model was measured by MTT assay after each of four storage treatments: storage at 24°C; storage at 4°C; cryoprotected with DMSO or cryoprotected with glycerol and storage in liquid nitrogen after freezing at different cooling rates to -80°C. The biological activity of 3DCS decreased 25% after 6 days of storage at 24°C and 80% at 4°C. In contrast, the biological activity of CD decreased to 60% at 24°C after six days, but remained about 95% at 4°C.
There was no effect of loading of 10% DMSO or glycerol in the 3DCS, but 15% of activity was lost in the CD. After freezing at different cooling rates (1-10°C/min) and storage in liquid nitrogen, the loss of activity was 40 - 70% in the CD and no loss in the 3DCS. When the 3DCS was cultured at 37°C after 4°C storage or cryopreservation, the biological activity did not recovered to the level of non-treated cells.
As shown in this study, cryopreservation of 3DCS kept biological activity compared to 4°C storage, but it is necessary to find better freezing conditions to maintain the biological activity of dermis.

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