Cryopreservation of Composite Tissue Transplants

The purpose of this study is to demonstrate the feasibility of the cryopreservation or composite tbsucs and to dctcrmine [he optimal parameters lor cooling, thawing. and perfusion of cryoprotectant. Composite tissue allotransplantation is a technique which holds great promise for reconstruction of severe upper extremity defects, but is limited by the scarcity of suitable donor parts. This shortage of tissue is expected to become more critical as the techniques of immunomodulation become less morbid and the indications for transplantation broaden. A reliable technique to preserve donor tissues long term could help meet this demand. In our laboratory, rat ovaries and testes have been successfully cryopreserved and transferred to recipient animals, and we believe the same techniques can be modified and used to eryopreserve composite tissue allotransplants. The rat epigastric flap model will be used as it is the simplest composite tissue which can be reliably transferred using microvascular technique. In the first phase of the study, 10 flaps will be harvested and perfused with the cryoprotective agent using the protocol established for organ preservation, then cooled to -85°C at varying rates. They will be kept frozen at -85°C for 14 days, and then thawed using the established protocol. MTT and Factor VIII assays will be performed to determine skin cell and endothelial cell viability, respectively. Tissue viability will be plotted against cooling rate to determine the optimal cooling rate. In the second phase of the study, rates of thawing will be varied while cooling is performed at the rate determined as optimal in phase I, to determine the optimal thawing rate. In the third phase perfusion with varying concentrations of cryoprotectant agent will be used prior to freezing and thawing at the predetermined optimal rates, to determine the optimal concentration gradient of cryoprotective agent.