目的 探索使用新型冻存剂进行人源脂肪组织冻存及移植的有效性。 方法 标本来源于2022年1至3月在北京大学第三医院成形外科进行吸脂术的健康成年女性，将获得的脂肪组织离心后随机分为9组，分别采用不同的冻存液[A组、B组、二甲基亚砜(DMSO)组]及冻存时长(1、2、3个月组)于液氮中冻存。A组冻存液组分为右旋糖苷40(DEX)＋氨基酸＋维生素＋无机盐，B组冻存液组分为DMSO＋DEX，DMSO组采用传统冻存液，组分及配比为10% DMSO＋20%胎牛血清(FBS)＋70% DMEM-12。采用5 ml冻存管，脂肪∶冻存液＝3∶2，每组冻存6管。脂肪组织复温后，采用HE染色观察组织学形态，采用免疫组化染色进行脂滴包被蛋白(Perilipin)定量分析，通过阳性细胞数量与总细胞数量的比值计算Perilipin阳性率；采用CCK-8法测定脂肪细胞活性。选择健康无特定病原体级裸鼠38只，根据移植脂肪使用不同的冻存保护剂(A组、B组、DMSO组)分为3组，每组各12只，另外2只作为day 0对照组，各组脂肪冻存时间均为3个月。裸鼠腹腔麻醉后，每侧背部注射复温后的冻存脂肪各0.9 ml，分别于移植后1、2、3个月通过MRI扫描及三维软件计算脂肪组织存留率并进行组间比较；小鼠处死后取移植的脂肪组织，采用HE染色和免疫组化染色进行组织形态学观察及Perilipin定量分析。采用GraphPad Prism 8.0软件对数据进行统计学分析，符合正态分布的计量资料以±s表示，整体多组间比较采用重复测量资料的方差分析，同一时间点组间数据比较采用Tukey多重检验。 结果 在液氮中冻存1～3个月后不同冻存液组的脂肪组织形态接近正常新鲜脂肪组织，各组Perilipin阳性率差异无统计学意义(P>0.05)；CCK-8法提示冻存1个月和3个月时DMSO组脂肪细胞活性优于A组和B组(P<0.01)，冻存2个月时DMSO组和B组脂肪细胞活性优于A组(P<0.01)。动物实验中冻存脂肪移植后1～3个月体积存留率各组之间差异无统计学意义(P>0.05)，移植1～3个月后各组脂肪组织出现不同程度局部坏死伴炎症反应，Perilipin阳性率各组之间差异无统计学意义(P>0.05)。 结论 使用新型冻存液冻存脂肪组织的效果与传统冻存液相比未见显著差异，且避免了使用DMSO或FBS作为冻存剂对人体潜在的不良作用，理论上更加安全。 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.
Evaluation of the efficacy of cryopreservation of human adipose tissue with novel cryoprotective agents
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.
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