Successful preservation of human skin by vitrification

Abstract

The vitrification technique was applied to the preservation of human skin. This technique was simple, and no expensive equipment was needed. Split-thickness human skins from 8 patients were immersed in vitrification solution for 10 minutes at room temperature, immediately plunged into a liquid nitrogen tank, and cryopreserved for 3 weeks. The vitrification solution consisted of 40% ethylene glycol (vol/vol) and phosphate buffered saline solution that contained 30% Ficoll 70 (vol/vol; Wako Junyaku, Co, Tokyo, Japan) and 0.5 mol/L sucrose. The viability of vitrified and cryopreserved skin was evaluated with the trypan blue dye exclusion test, the methyl-thiazoldiphenyl-tetrazolium (MTT) colorimetric assay, and a culture test of the keratinocytes obtained from vitrified skin. The results of the trypan blue dye exclusion test showed 87.4% of viable cells, and MTT developed an average 0.817 absorbance. When vitrified skin was compared with 4 degrees C refrigerated skins after 3 weeks of storage, the difference of viability was significant both on the trypan blue dye exclusion test (P < .05) and on the MTT assay (P < .01). However, there was no significant difference in the viability of vitrified skins compared with fresh skin. Furthermore, keratinocytes from vitrified skin grew uneventfully in culture test. We used these vitrified skin allografts for patients with flame burns and electric burns. These allografts took well in both cases and promoted wound healing. We concluded that the vitrification method for skin preservation is simple and reliable, and this method could contribute to skin banking.