The Basic Study On Cytological Changes Of Adipose Tissue After Different Cryopreservation Time

Backgroud In 1893, it was reported that autologous fat transplantation for the first time to repair the defect of soft tissue. But the high absorption rate of the transplanted recipient area is a major challenge for both of doctors and patients. The main reason for the high absorption rate of autologous fat transplantation may be due to the necrosis of the adipose tissue after transplantation. This high absorption rate necessitates overcorrection and reinjection procedures in the desired area, which are associated with repeated suction-assisted lipectomy operations to obtain the fat, as well as higher cost, unfavorable appearance, increasing patient morbidity, and discomfort. Therefore, in order to solve this problem, it is necessary to find a long-term cryopreservation of the obtained fat, which is essential to both doctors and patients. In 1990, Forunier first reported the cryopreservation of adipose tissue. Then many scholars have carried out a series of experiments and clinical research on autologous fat,including the cryopreservation of adipose tissue, adipose cells and adipose derived stem cells. Some researchers have also reported that transplantation of frozen fat caused less swelling and discoloration than fresh fat in the operation area. Researchers have done a lot of improvement and optimization,such as optimization of the method of obtaining fat before freezing;Control the freezing rate and add the freeze protection agent;Using the frozen container such as commonly used refrigerator(-20?), deep low temperature refrigerator(-80?),liquid nitrogen(-196?)for the cryopreservation. Due to the remove difficulties and toxicities of the cryoprotective agents,there are no reports about the transplantation of the adipose which has been add the cryoprotective agents. It was reported that no or very few harmful effects were generated on the fat samples cryopreserved at-20 ?without controlling of the cooling process and even without CPA, and live adipocytes were found after preservation at this temperature, and it also has been successfully applied in clinic. In decades, many scholars have done a great deal of research on the frozen adipose tissue and the optimization of the freezing method.However,inconsistent or even opposite results can be found in the literature, and there is no standard theory for the formation of frozen fat. Currently, on the basic study on cytological changes of adipose tissue after different cryopreservation time is also rarely reported.Objective The study was based on the absence of any cryopreservation solution and cryoprotective agents. After purification and centrifugation, the human adipose tissue was frozen in the refrigerator(- 20 ?). To observe and analyze the changes of cell metabolism, cell viability, and other aspects of adipose tissue after thawing. Preliminary exploring the cytological changes of adipose cells, optimization and standardization of the method for the adipose cryopreservation. Provided solid theoretical and experimental basis.Methods Extraction of human adipose tissue after isolation and purification contained a small amount of normal saline. Frozen for 1month, 2 months, 3 months, 6 months, 9 months, 12 months, 24 months in the refrigerator(- 20 ?). Then, compared with the fresh adipose tissue after thawing. Through hematoxylin-eosin staining,trypan blue staining,methods of flow cytometry oil,red O staining and glucose transfer method, investigating changes of cell vitality and the histological changes of human granular fat tissue, compare with fresh adipose tissue mixed with a small amount of normal saline.Results Adipose tissue particles morphology observation showed color from bright yellow and gradually fades, oil impurities increased gradually. Hematoxylin-eosin staining showed cryopreserved adipose tissue particles(frozen for 1, 2 months later)the tissue structure was clear and complete, with no significant difference between fresh fat.Frozen for 3, 6, 9 months later, although the existence of tissue structure, but the quality and quantity of cells decreased gradually. Freezing time is 12, 24 months later, the morphology and structure of tissues and cells have been severely damaged. Analysed by trypan blue staining and quantitative cell counting instrument, the results show: compared with fresh fat particles, particles with the increase of adipose tissue freezing time, the survival rate decreased gradually;frozen for 1, 2 months later the survival rate decrease is not obvious,the survival rate have no significant difference from the control group(P>0.05); frozen for 3 months later, the survival rate began to decline,and the survival rate have obvious significant difference from the control group(P<0.05); When the freezing time was increased to 6months, the survival rate decreased significantly. And with the increase of freezing time, the survival rate gradually decreased; until frozen for 24 months later, the survival rate was 3.30% + 1.54%(P<0.05). Flow cytometry showed that frozen for 1, 2 months later adipose tissue particles compared with the control group, the apoptosis ratio of no significant difference(P > 0.05); when frozen storage time was increased to 3 months, the apoptosis ratio has significantly decreased. and with the extension of cryopreservation time, the proportion of apoptotic cells gradually increased. Frozen for 24 months later, apoptosis proportion was 94.2% + 4.55%(P < 0.05). Oil red O staining displayed that intracellular lipid droplets of fresh fat particles was mostly red; Frozen for 1, 2 months later the red intracellular lipid droplets of adipose tissue have no significant differences from the control group;When the freezing time increased to 3 months,the number of red lipid droplets decreased and fusion into large lipid droplets.With the increase of cryopreservation time, red lipid droplets and blue stained nuclei number decreased significantly.Most are big red lipid droplet; To the freezing time of 24 months, the red intracellular droplet of adipose tissue is rare.Glucose transfer experiment results showed,assuming fresh fatty tissues of granule cell activity was 100%,that frozen for 1, 2 months later adipose tissue particles cell viability was 94.7%±4.40% and 92.5%±5.10%, and have no significant difference from the control group(P>0.05). When the freezing time increased to 3 months. The cell activity decreased significantly to 75.3% + 4.80%, and there was significant difference from the control group(P<0.05). With freezing time increased, the cell activity gradually decreased.Until frozen for 24 months later, the survival rate was 4.9%±3.20%.Conclusion Human adipose tissue particles purified under-20 ?frozen within 2 months have no significant difference between fresh adipose tissue particles in the survival rate of fat cells.