CRYOPRESERVATION OF VIABLE HUMAN SKIN SUBSTITUTES

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