Cryopreservation of composite tissues and transplantation: preliminary studies.

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5M dimethyl sulfoxide (Me(2)SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to -140 degrees C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n=10), perfused (n=8), and cryopreserved (n=17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n=10) was 11.17+/-2.01, which was not significantly different from fresh controls (n=10, 12.15+/-1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.

Cryopreservation of composite tissue transplants.

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.

Cryopreservation of composite tissue flaps: experimental studies in the rat.

Cryopreservation has the potential to improve availability of donor parts for composite tissue allotransplantation and may reduce their antigenicity. This study investigates whether the component tissues of composite flaps remain viable after cryopreservation. Forty-one epigastric flaps were harvested from Lewis rats. Twenty-one flaps were perfused with DMSO/trehalose, frozen by controlled cooling to -140 degrees C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylin & eosin and factor VIII staining. An epithelial viability index was calculated for 10 fresh and 11 cryopreserved flaps using the MTT assay. In all cryopreserved samples, hematoxylin & eosin, and factor VIII staining revealed a well-preserved cellular architecture, which was indistinguishable from fresh specimens. The viability index for the cryopreserved samples was 10.90 +/- 2.09 compared with 12.15 +/- 1.32 for fresh flaps (P = 0.123). Results suggest that the skin, adipose, and vascular endothelial cells of composite tissue flaps retain their viability after cryopreservation and thawing.