Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.