Reduced Engraftment and Wound Closure of Cryopreserved Cultured Skin Substitutes Grafted to Athymic Mice

Cryopreservation of cultured skin substitutes is a requirement for establishment of banks of alternative materials for treatment of acute and chronic skin wounds. To determine whether cryopreservation of skin substitutes that contain cultured cells reduces their efficacy for wound closure, cell–biopolymer grafts were frozen, recovered into culture, and grafted to wounds on athymic mice. Grafts consisted of cultured human keratinocytes and fibroblasts attached to collagen–glycosaminoglycan substrates that were frozen in cell culture medium with 20% serum and 10% DMSO at a controlled rate and stored overnight in liquid nitrogen. After recovery into culture for 24 h, frozen or unfrozen (control) skin substitutes were grafted to full-thickness wounds on athymic mice. Wound area and surface electrical capacitance were measured at 2, 3, and 4 weeks after grafting at which time animals were sacrificed. Wounds were scored for presence of human cells by direct immunofluorescence staining with a monoclonal antibody to HLA-ABC. The data demonstrate that cell–biopolymer grafts are less efficacious after controlled-rate cryopreservation using 10% DMSO as a cryoprotectant. Frozen grafts at 4 weeks after surgery have significantly smaller wound areas, higher capacitance (wetter surface), and fewer healed wounds that contain human cells. The results suggest that these conditions for cryopreservation of cultured grafts reduce graft viability. Improved conditions for cryopreservation are required to maintain viability and efficacy of cultured skin substitutes after frozen storage.