Cryopreservation of composite tissue transplants

Abstract

Composite tissue allotransplantation holds great promise for upper extremity reconstruction but is limited by donor part availability. Cryopreservation may increase the availability of donor parts and even reduce antigenicity. The purpose of the study was to evaluate the viability of cryopreserved composite tissues and to demonstrate the feasibility of microvascular isotransplantation of cryopreserved composite flaps. Twenty epigastric flaps were harvested from Lewis rats. Ten flaps were analyzed fresh. Ten flaps were perfused with dimethyl sulfoxide (DMSO)/trehelose cryoprotectant agent (CPA), frozen by controlled cooling to -140 degrees C, and stored for 2 weeks. Flaps were evaluated by factor VIII endothelial staining and MTT tetrazolium salt assay. For the in vivo phase, 30 flaps were harvested. Ten were transplanted fresh to isogenetic recipient animals, ten were perfused with CPA and transplanted, and ten were cryopreserved for 2 weeks, thawed, and transplanted. All cryopreserved samples displayed intact vascular endothelia on factor VIII staining. On MTT analysis, the epithelial viability index for the cryopreserved samples was not significantly different from fresh controls (p = 0.12). All freshly transplanted flaps (10/10) were viable at 60 days. Nine of ten flaps in the perfused/transplanted group were viable at 60 days. Survival of cryopreserved/transplanted flaps ranged from 5 to 60 days. The skin and vascular endothelial components of composite tissue flaps appear to retain their viability after cryopreservation. The in vivo studies demonstrate that the long-term survival of cryopreserved composite tissue transplants is feasible and support an indirect injury, rather than direct injury from freezing or cryoprotectant agents, as the mechanism of flap loss.

Figure 1

(Left) Schematic of donor surgical procedure, showing flap design. (Right) Showing method of epigastric flap harvest, including a cuff of the femoral vessels.

Figure 2

(Above, Left) Isotransplantation recipient animal under anesthesia with outline of defect marked. (Above, Right) After creation of abdominal defect and preparation of recipient vessels. (Below, Left) Microscopic view of vascular anastomosis. (Below, Right) Immediately after isotransplantation of a cryopreserved and thawed epigastric flap.

Figure 3

Schematic of the two-pump system for providing graded perfusion of cryoprotective agent.

Figure 4

(Above, Left) Histologic section of a cryopreserved/thawed flap vessel and surrounding connective tissue with H&E staining, showing preservation of cellular architecture. (Above, Right) High-power view of the perivascular region of a cryopreserved/thawed flap with H&E staining. (Below, Left) Histologic section of cryopreserved epigastric artery with factor VIII staining, showing intact vascular endothelium. (Below, Right) High-power view of the cryopreserved epigastric artery, with factor VII endothelial staining.

Figure 5

(Left) Recipient animal on postoperative day 1 after isotransplantation of a cryopreserved and thawed epigastric flap. (Center) Appearance of transplant on postoperative day 7, with a small area of peripheral necrosis. (Right) Appearance of transplant on postoperative day 60, with normal color and hair growth.