Cryopreservation of viable human skin substitutes

Abstract

The present invention relates generally to systems and methods for preparing, storing, shipping and using skin equivalents made by organotypic culture. In particular, the present invention relates to systems and methods for cryopreserving viable skin substitutes.

We claim: 
     
         1 . A kit comprising:
 a cryopreserved organotypically cultured skin equivalent;   a diffusion mediator; and   a tissue compatible solution,   wherein the cryopreserved organotypically cultured skin equivalent is equilibrated with a cryoprotectant and substantially free of excess cryoprotectant on the exterior surface of the cryopreserved organotypically cultured skin equivalent.   
     
     
         2 . The kit of  claim 1 , further comprising a sealable enclosure, wherein the cryopreserved organotypically cultured skin equivalent is packaged in the sealable enclosure in the absence of additional cryoprotectant to provide a packaged organotypically cultured skin equivalent. 
     
     
         3 . The kit of  claim 2 , wherein the sealable enclosure is a sterile bag. 
     
     
         4 . The kit of  claim 3 , wherein the sterile bag is further enclosed in a second bag. 
     
     
         5 . The kit of  claim 4 , wherein the second bag is a plastic, foil, or Mylar bag. 
     
     
         6 . The kit of  claim 2 , further comprising a culture vessel, wherein the cryopreserved organotypically cultured skin equivalent is provided in the culture vessel packaged in the sealable enclosure. 
     
     
         7 . The kit of  claim 6 , wherein the culture vessel comprises an insert having a bottom planar surface formed from a porous membrane. 
     
     
         8 . The kit of  claim 2 , wherein the cryopreserved organotypically cultured skin equivalent is prepared using the method comprising:
 treating an organotypically cultured skin equivalent in a cryoprotectant solution in a single step, wherein said organotypically cultured skin equivalent comprises stratified squamous epithelia on a dermal layer comprising fibroblasts embedded in collagen, and wherein the cryoprotectant solution comprises glycerol in an amount that is about 21% to about 45% of the cryoprotectant solution by volume;   separating the treated organotypically cultured skin equivalent from excess cryoprotectant solution to provide a treated organotypically cultured skin equivalent that is substantially free of excess cryoprotectant on the exterior surfaces of the treated organotypically cultured skin equivalent; and   freezing the packaged organotypically cultured skin equivalent to provide the cryopreserved organotypically cultured skin equivalent that retains viability after thawing.   
     
     
         9 . The kit of  claim 8 , wherein the cryoprotectant solution comprises glycerol in an amount that is about 25% to about 40% of the cryoprotectant solution by volume. 
     
     
         10 . The kit of  claim 8 , wherein the cryoprotectant solution comprises glycerol in an amount that is about 32.5% of the cryoprotectant solution by volume. 
     
     
         11 . The kit of  claim 8 , wherein the cryoprotectant solution comprises glycerol in an amount that is about 37.5% of the cryoprotectant solution by volume. 
     
     
         12 . The kit of  claim 1 , wherein the cryopreserved organotypically cultured skin equivalent is stored at temperatures from about −50° C. to about −100° C. 
     
     
         13 . The kit of  claim 12 , wherein cryopreserved organotypically cultured skin equivalent is able to be stored from about 1 to 24 months without a substantial loss of viability. 
     
     
         14 . The kit of  claim 1 , wherein the diffusion mediator is selected from the group consisting of an absorbent medium, a porous membrane, and a dialysis bag. 
     
     
         15 . A kit of  claim 14 , wherein the diffusion mediator is an absorbent medium and the cryopreserved organotypically cultured skin equivalent is placed on the absorbent medium. 
     
     
         16 . The kit of  claim 15 , wherein the absorbent medium is selected from the group consisting of Telfa pads, foam pads, gauze pads, and cellulosic pads containing the tissue compatible medium. 
     
     
         17 . The kit of  claim 1 , wherein the tissue compatible solution is a buffered solution. 
     
     
         18 . The kit of  claim 17 , wherein the buffered solution is selected from the group consisting of DMEM, Ham's F-10, Ham's F-12, DMEM/F-12, Medium 199, MEM, RPMI, HEPES, Tris, MOPS, and Trizma buffers. 
     
     
         19 . The kit of  claim 17 , wherein the buffered solution is provided at an amount to provide a pH in the range of 7.0 to 7.4