Cryopreservation of viable human skin substitutes

A method of producing a cryopreserved skin equivalent comprising:

• providing a culture dish comprising an insert movable between upper and lower positions in said culture dish, said insert having a bottom planar surface formed from a porous membrane,
• forming a dermal equivalent comprising fibroblast cells on said porous membrane in said insert, wherein said insert is placed in said upper position in said culture dish,
• culturing said fibroblast cells to form a dermal equivalent,
• applying keratinocytes to said dermal equivalent,
• culturing said keratinocytes in a culture medium under conditions such that said keratinocytes form a skin equivalent comprising stratified epithelium,
• removing said culture medium,
• lowering said insert to said lower position,
• treating said skin equivalent with a cryoprotectant solution wherein the cryoprotectant solution comprises glycerol in an amount that is about 21% to about 70% of the cryoprotectant solution by volume,
• separating the treated organotypically cultured skin equivalent from excess cryoprotectant solution to provide a treated skin equivalent that is substantially free of excess cryoprotectant on the exterior surfaces of the skin equivalent, and
• freezing said skin equivalent in said culture dish in the absence of additional cryoprotectant to provide a cryopreserved skin equivalent that retains viability after thawing.