The Protective Effects Of Antioxidants To Cryopreserved Human Granular Adipose Tissue

Objective Feasibility analysing of human granular adipose tissue changes in cell structure and viability under the current commonly used antioxidant added into cryoprotectant after thawing, for the aim of clinical implementation, "repeated use with once cryopreserved" and improve survival rate of human granular fat tissue after transplantation and clinical satisfaction, reduce the pain of patients and the workload of doctors, provided solid theoretical and experimental basis.Methods Choose the several kinds of antioxidant current commonly used,superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and vitamin E(VE), adding into the stock solution which basis of preparation permeable vitrification cryoprotectants fluid(saline PS + 8% dimethyl sulfoxide DMSO)respectively, than put in-4 ℃ refrigerator frozen storage, rewarming after 2months after mixed with purifying human granular fat tissue, through hematoxylin- eosin staining, oil red O staining and ROS assay kit, methods of flow cytometry, investigating changes of cell vitality and metabolic of human granular fat tissue, compare with human granular fat tissue without adding antioxidants cryopreserved.Results Hematoxylin-eosin staining showed ununiformed structure, large cyst-like lipid pool and necrotic area of the human granular adipose tissue grafts cryopreserved with basic cryoprotectant medium composed of physiological solution and 8% dimethyl sulfoxide. Uniformed structure, reduced large cyst-like lipid pool and necrotic area were observed under microscope in groups treated with basic cryoprotectant medium supplemented with different antioxidants,superoxide dismutase(100-600 IU/m L), reduced glutathione(1-15 mmol/L), and tocopherol(vitamin E, VE, 1-2.5 mg/m L). Oil red O staining displayed that anumber of lipid droplets were ruptured in human granular adipose tissue grafts cryopreserved with conventional cryoprotectant medium, and the number of adipocytes counted with hematoxylin stained-nucleus was also decreased.Intracellular droplets of human granular adipose tissue were increased in groups of cryopreserved with conventional cryoprotectant supplemented with antioxidants. The data assayed by reactive oxygen species kit indicated that mean fluorescent intensity of adipocytes was significantly decreased in human granular adipose tissue cryopreserved with conventional cryoprotectant supplemented with three kinds of antioxidants compared with group treated with solely conventional cryoprotectant(P<0.05). Flow cytometry analysis demonstrated that apoptosis rate of adipocytes in human granular adipose tissue cryopreserved with supplementation of antioxidants was significantly lowered than that cryopreserved with conventional cryoprotectant(PS+8%DMSO)(P<0.05).Conclusion 1. The structure integrity, lipid droplet and cell survival of human granular adipose tissue graft are impaired when cryopreserved at-4 ℃ with conventional cryoprotectant, physiological solution containing 8% dimethyl sulfoxide. 2. Supplementation of antioxidants, superoxide dismutase, reduced glutathione or tocopherol(vitamin E) ameliorates the freeze thawing impairment to human granular adipose tissue assessed in cell viability, droplet content and antioxidative capacity. 3. The protective effects of antioxidants to cryopreserved human granular adipose tissue from freeze thawing injury display a dose-effect dependent manner.