Harriger M D, Supp A P, Swope V B, Boyce S T
Shriners Burns Institute, University of Cincinnati, Cincinnati, Ohio, USA.
Cryobiology. 1997 Sep;35(2):132-42. doi: 10.1006/cryo.1997.2030.
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.
培养皮肤替代物的冷冻保存是建立用于治疗急慢性皮肤伤口的替代材料库的一项要求。为了确定含有培养细胞的皮肤替代物冷冻保存是否会降低其伤口闭合功效，将细胞 - 生物聚合物移植物冷冻，复苏后培养，然后移植到无胸腺小鼠的伤口上。移植物由附着在胶原 - 糖胺聚糖基质上的培养人角质形成细胞和成纤维细胞组成，这些基质在含有20%血清和10%二甲基亚砜（DMSO）的细胞培养基中以可控速率冷冻，并在液氮中储存过夜。在复苏培养24小时后，将冷冻或未冷冻（对照）的皮肤替代物移植到无胸腺小鼠的全层伤口上。在移植后2、3和4周测量伤口面积和表面电容，此时处死动物。通过用抗HLA - ABC单克隆抗体进行直接免疫荧光染色对伤口中人类细胞的存在情况进行评分。数据表明，使用10% DMSO作为冷冻保护剂进行可控速率冷冻保存后，细胞 - 生物聚合物移植物的功效降低。术后4周时，冷冻移植物的伤口面积明显更小，电容更高（表面更湿润）且含有人类细胞的愈合伤口更少。结果表明，这些培养移植物的冷冻保存条件降低了移植物的活力。需要改进冷冻保存条件以在冷冻保存后维持培养皮肤替代物的活力和功效。
