Experimental Study On The Characteristics Of Human Adipose Stem Cells After Different Cryopreservation Periods

Objective:The purpose of this project is to study the effect of extracting adipose stem cells from adipose tissue after different cryopreservation times to differentiate into mature adipocytes after passage in vitro,and to test the ability of adipogenic stem cells to differentiate into adipogenic and secretory functions for human adipose stem cells.The preservation of biological characteristics and its application potential provide a certain basis.Methods: 6 cases of adult female abdomen or thigh fat were extracted and separated and purified,and each had 20 ml purified fat.A portion of the fat was frozen with trehalose as a cryoprotectant for 3 months,and rewarmed after 6 months.Fresh adipose tissue and frozen fat-derived adipose stem cells were cultured in a culture flask to complete culture medium for the third generation.The third-generation adipose stem cells were cultured into mature adipocytes in a 6-well plate using medium containing dexamethasone,insulin,indomethacin,and isobutylmethylxanthine(IBMX)and insulin-containing medium in that order.The growth curve of adipose stem cells in different groups was determined by MMT method,adipogenic differentiation ability was identified by oil red staining,surface antigen characteristics were detected by flow cytometry,and vascular endothelial growth factor(VEGF)in the 3rd generation adipose stem cell culture medium was determined by ELISA.The contents of hepatocyte growth factor(HGF)and basic fibroblast growth factor(b FGF)were studied to investigate their proliferation,adipogenic differentiation,and secretory functions.Results:(1)The adipocytes extracted from the three groups were observed to have a few polygonal adherent cells one day after culture,and these cells were gradually transformed into long spindle-shaped cells.After reaching 80% confluence,cells were observed to be spindle-shaped and arranged in a parallel or swirling order.There was no significant difference in the growth curve of adipose stem cells between the three groups.(2)The three groups of adipose stem cells can be gradually seen in the conditioned medium,the cells gradually become shorter,round,the small visible bright lipid droplets in the cytoplasm,then gradually increase the size of the lipid droplets,fusion,to about 14 days mature fat cells At this time,the cell volume becomes larger,visible larger lipid droplets,and some even occupy more than 80% of cells.(3)Flow cytometry showed that adipose-derived stem cells still expressed high levels of CD44 and CD90 after freezing,consistent with the characteristic phenotype of mesenchymal stem cells.Adipose stem cells still have the characteristic phenotype of mesenchymal stem cells after cryopreservation.(4)After 3 groups of adipose-derived stem cells were passed to the third passage and grew to 80% confluence,the vascular endothelial growth factor(VEGF),hepatocyte growth factor(HGF),and basic fibroblast growth factor were measured by ELISA.b FGF)content,VEGF content between the groups P> 0.05,no significant difference;HGF content between the groups P> 0.05,no significant difference;b FGF content between the groups P> 0.05,no significant difference.Conclusion:Human adipose-derived stem cells within 6 months of cryopreservation still have stable biological properties,and they can differentiate into mature adipocytes under the induction of orientation in vitro,and can be used to assist fat transplantation.