In order to establish optimal condition for long-term artificial skin preservation, kertinocytes, fibroblasts and melanccytes were cultured from normal rats or human skin.
Using therse cultured cells and collage gel, epidermal sheets, artificial dermis, and artificial skin were prepared in vitro. The three types of single cells and artificially constructed tissues were frozen with different concentrations, two
types
of
cryoprotective agents, and at different freezig speeds. After freezing-thawing in each combination, the viability of the single cells and tissues was checked by the trypan blue dye exclusion methods and the following results were obtained.
1) Viability of the single keratinocytes was almost never influenced by the freezing rate or type of cryoprotective agent.
2) Viability of the cultued epidermal sheets was similar to that of the single kerationocytes.
3) Viablity of the melanocytes was greatly influenced by the freezing rate and the type of cryoprotective agent. High survival was observed in dimethyl-sulfoxide(DMSO) and viability was in inverse to the freezing speed.
4) Viability of the single fibroblasts was influenced by the freezing rate rather than the type or concentration of cryoprotective agent.
5) Viability of th artificial dermis depended on the concentration method, and the viaility of the fibroblast in the uni-directionally contracted artificial dermis was similar to that of the single fibroblast.
6) Taken as a whole, the optimal freezing conditions satisfying each type of singel cell and artificially constructed tissue were selected: 1.5M DMSO as an optimal conentration and type of cryoprotective agent, and 1℃/minute as the optimal
freezing
rate. in these freezing conditions, the viability of each type of single cell and artificially constructed was maintained up to 80%, and the artificial skin which were freeze-thawed in these freezing conditions were grafted successfully in the
animal
experimental model. These facts suggest that optimal freezing conditions may be used in human artificial skin preservation.
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