Transplantation of cryopreserved cultured epidermal allografts

To optimize cryopreservation methods for cultured epidermal allografts (CEAs) for transplantation onto wounds.

Conditions were determined to optimize the cryopreservation of CEAs. Cryopreserved CEAs were then grafted onto 16 donor sites in a double blind randomized trial.

CEAs were grown in culture. Viability of cryopreserved CEAs was determined by: 1) trypan blue dye exclusion; 2) histology; 3) flow cytometry; and 4) acid lipase activity. Cryopreserved CEAs were grafted onto 16 donor sites. Wound healing assessments included 1) visual assessment of healing; 2) histologic assessment of re-epithelialization and differentiation; and 3) visual scar assessment.

CEAs were cryopreserved using a controlled rate freezer with an experimentally determined setting of -7 degrees C for the CEA freezing point. Such freezing conditions resulted in retention of approximately 92% of the original viability, no loss in basal keratinocytes as determined by flow cytometry, and no change in acid lipase activity. CEAs cryopreserved according to this method were grafted onto 16 donor sites in a double blind randomized trial. All donor sites underwent complete healing. Histologic examination of biopsies taken from the center of the wound beds showed that CEA-treated wounds significantly accelerated the rate of re-epithelialization (7.8 +/- 0.6 days vs. 9.2 +/- 0.9 days for CEA- and control-treated wounds, respectively; p = 0.039) and epithelial differentiation (p = 0.023) compared with control sites. Longterm results showed that CEA-treated wounds were comparable to control sites with regard to: 1) pigmentation; 2) scar height; 3) scar pliability; 4) vascularity; and 5) pain. Wounds treated with cryopreserved CEAs remained durable and not prone to blistering after healing.

CEAs can be successfully cryopreserved for long-term storage. Upon retrieval from storage, CEAs may be used to treat partial thickness wounds.