| Objective:To explore the effect of different frozen storage conditions on human adipose cell vitality,seek a more satisfactory granular fat frozen storage condition,and provide a theoretical basis for autologous fat transplantation in clinical.Methods : The fat granule samples from interior thigh of 41 patients who underwent conventional liposuction were collected respectively,and then the freeze protection cryogenic liquid containing 60% fetal bovine serum + 25% dulbecco modified eagle medium(DMEM)+ 15% dimethyl sulfoxide(DMSO)were added into them and cryopreserved after washed 2 times by physiological saline rinse and placed quiescently to be separated.Three fat copies of the specimen were stored at common freezer of-20℃,cryopreservation refrigerator of-80℃,and liquid nitrogen freeser of-196 ℃,respectively.The samples that had been frozen for 3 and 6 months were selected to tested about the activity of frozen fat after rewarming.The morphology of cryopresered granule fat cells were observed by hematoxylin-eosin(H&E)staining and trypan-blue staining was used to calculate the death cell percentage.The creatine kinase level was tested to calculate fat cell failure rate.The CD105 on adipose derived stem cell was detected by immunohistochemical methods to compare the effect of different frozen storage temperatures and frozen storage time on the fat cell activity in vitro.A total of 6 nude mice were used to establish the fat transplantation model.The fat granular samples that had been frozen at-20℃,-80℃,-196 ℃ for 6 months were injected respectively into the subcutaneous area of one side of mice back.The mice were sacrificed at 4 weeks after the procedure,and the grafted fat tissues were harvested.The extracted fats were evaluated by photographic analysis,volume measurements,histological examination and transmission eleclron microscopy.All data were analyzed by SPSS 17.0 statistical package.Results:1.The fat cells vitality was tested in vitro:(1)The survival rates of fat cells were assessed by trypan-blue staining analysis.The result showed that the fat cell survival rates in the groups stored at-20℃,-80℃ and-196℃ after 3 months were 18.79±2.51%,86.21±1.51% and 90.05±2.77%;8.67±1.83%,81.34±2.39% and 89.31±2.65% in the groups after 6 months,respectively.Statistical analysis showed that the data compared among groups and between the groups of-20℃ and-80℃ were all of statistical significance(P <0.05).The fat cell survival rates under the same frozen storage time in the groups stored at-20℃,-80℃ and-196℃ increased gradually depending on the debased temperature.The number of viable fat cells frozen at-196℃ was maximum in three temperature groups and the fewest in the group cryopreserved at-20℃(Ρ<0.05).Under the same frozen storage temperature,the fat cell survival rates in the groups stored at-20℃,-80℃ for 3months were more higher than ones in the 6 months groups.But there were no obvious differences between the-196℃ groups under the same frozen storage time in the fat cell survival rates(Ρ>0.05).(2)The determination of creatine kinase showed that the enzyme activities in the groups stored at-20℃,-80℃ and-196℃ after 3 months were 0.042±0.003,0.072±0.002,and 0.081±0.002;0.038±0.002,0.063±0.002,and 0.079±0.003 in the groups after 6 months,respectively.The enzyme activities in the groups cryopreserved at-20℃ and-80℃ for 3 months were higher than the ones for 6 months(Ρ<0.05),but there were no obvious differences between the-196℃ groups under the same frozen storage time in the enzyme activities(Ρ>0.05).The enzyme activity of the-196℃ group was the highest,the lowest in the-20℃ group and mediate in the-80℃ group under the same frozen storage time(Ρ<0.05).(3)The comparative observation for the fat cell morphology by H&E stainingunder the microscope found that the cell membrane was ruptured,network connection partly interrupted,the nucleus observed occasionally in the group cryopreserved at-20℃ for 3 months.However,the cell membrane was ruptured more seriously,network connection interrupted thoroughly,the nucleus almost disappeared with liquefaction necrosis and cyst cavity in the group cryopreserved at-20℃ for 6 months.The fat cell morphologies got more improved in the group cryopreserved at-80℃ for 3 and 6months than the one in group cryopreserved at-20℃ for the same period and kept well in the groups at-196℃ for 3 and 6 months.Immunohistochemical staining of CD105 showed that there were not ADSCs in-20℃ groups and only a few ADSCs in the groups at-80℃ and-196℃.2.The model establishment of nude mice grafted granule fat:The 0.2ml samples of granule fat stored at-20℃,-80℃ and-196℃ for 6 months were injected subcutaneously into the 6 nude mice backs respectively.The mice were sacrificed at 4weeks after the procedure,and the grafted fat tissues were harvested respectively.The resorption rates of granule fat stored at-20℃,-80℃ and-196℃ for 6 months 4 weeks after transplantation were 90%,65% and 55%,respectively.The comparative observation for the grafted fat cell morphology by H&E staining under the microscope found that the grafted granule fat lacked the normal fat lobule structure,showed more foam change in them and ruptured membrane in the group at-20℃.The well cell survival,integrated fat lobule structure,steady cell morphology and part of the vascular ingrowth were shown in the another two groups.The cell ultrastructure observation by transmission electron microscope for the grafted granule fat 4 weeks after transplantation showed that there were many neonatal adipocytes in all groups,but the neonatal adipocytes were more mature in groups at-80℃ and-196℃ and the most newborn capillaries in the group at-196℃.Conclusion:1.The use of the cryopreserved adipose tissue for transplantation is feasible.2.The fat cells vitality of groups at the temperature of-196℃ are superior to ones at-80℃ and-20℃.3.The fat cells can be cryopreserved at-196℃ for 6 months,or even longer time,which is the ideal frozen storage temperature.The fat cells vitality at-80℃ for 6 months is lower than one at-80℃ for 3 months.So that the-80℃ can be used for fat short-term storage.It is not recommended to choose-20℃ to cryopreserve the fat tissue. |
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