Skin banking: a simple method for cryopreservation of split-thickness skin and cultured human epidermal keratinocytes

A simple unit has been developed for the simultaneous passive cooling of small and large amounts of allograft or autograft split-thickness skin, as well as cultured human epidermis. An expanded polystyrene box of variable size, aluminum plates, and cellulose tissue are fused. This unit is cooled in a -70 degrees C constant-temperature mechanical refrigerator. Maximal cooling rates of -1.3 degrees C min-1 are obtained in a box with a constant wall thickness of cellulose tissue. The cooling rate can be varied by altering the number of cellulose layers. Exothermic temperature plateaus associated with skin cooled in this unit last for less than 0.3 minutes. The viability of the cryopreserved skin was determined by using up to four methods simultaneously: a dye-exclusion test with trypan blue; glucose consumption; production of lactate; and carbon-13 nuclear magnetic resonance spectroscopy. Using a cryoprotectant medium with 15% (vol/vol) glycerol for split-thickness skin 0.25 mm thick, a storage time of up to 509 days at -70 degrees C was observed, with only a small decrease in viability (trypan blue, 62.5%; glucose consumption, 71 to 90% compared with freshly harvested skin). Storage in liquid nitrogen did not significantly improve results (p greater than 0.05).