Experimental Study On The Effect Of Trehalose On Activity Of Post-cryopreserved Adipocyte

Background: Autologous fat transplantation was invented over a century ago,and it has been widely used for many years thanks to many advantages and a good plasticity of it,and it is also a focus of research.The important and difficult points of research have always lied in the maintenance of survival rate of post-transplanted fat.There have been many studies and reports that improvements have been achieved in terms of fat donor site,method of extraction,extraction and suction tool,method of purification and addition of many cytokines,although a certain effect has been achieved,the fat absorption cannot be avoided.Through multiple comparisons,we believed that the best solution was multiple transplantations.However,the solution that fresh fat is used as a donor each time is accompanied by multiple liposuctions,accordingly,the patient has to suffer from more pains and is at a higher risk of surgical complications.Sufficient fats were extracted by the first liposuction,as for the subsequent transplantation,the use of cryopreserved fat for transplantation was an effective solution,as a result,the protection of activity of post-cryopreserved fat has become a focus of research.In recent years,there have been many researches concerning the fat cryoprotectant,dimethyl sulfoxide(DMSO),glutathione peroxidase,fetal calf serum,polyvinylpyrrolidone(PVP),maltose and methylcellulose(MC)are all believed to be able to improve the survival rate of post-cryopreserved adipocyte.However,these substances can be hard to remove after being mixed with adipocyte,part of them even have cytotoxicity.Seeking for a clinically-applicable cryoprotectant is a method that enables us to promote and popularize the “one liposuction and multiple transplantations”.Objective: To discuss and explore the effect of trehalose as a cryoprotectant on the cell activity of post-cryopreserved fatty tissue through the observation of general appearance of post-cryopreserved fat,HE staining,oil red O staining,detection of glucose transfer amount and creatine kinase.Methods: The adult females were performed with a suction of fat particle at abdomen or thigh,the range of suction of fat was designed,20 ml syringe was used to extract the subcutaneous fat after a tumescent anesthesia was performed locally,then the extracted fat was centrifuged for 2 min at 800 rpm and purified,the upper-layer grease and lower-layer mixed liquor were discarded,the middle-layer granule fat was taken and divided into 3 groups: group A(trehalose group),group B(fetal calf serum+ 10% DMSO)and group C(normal saline group).After 12 weeks of cryopreservation in liquid nitrogen,a fast water-bath rewarming was performed,the survival of adipocyte was observed through the observation of general appearance of post-cryopreserved fat,HE staining,oil red O staining,detection of glucose transfer amount and creatine kinase.Results:The configuration of fat in group A and B were found to be good by the observation of general appearance of post-cryopreserved fat,which were yellow in color,while the organizational structure of fatty tissue in group C was loose and inclined to be light in color.It was found by HE staining that,the fatty tissue from group A and B was clearly structured and orderly arranged,the cell nucleus was visible;while the fatty tissue from group C was disorderly structured and found to have areas of necrosis.The result of detection of glucose transfer amount of group A,B and C was respectively 0.61±0.12mmol/L?0.64±0.062mmol/L and 0.47±0.09mmol/L.the result of determination of activity of creatine kinase of three groups was respectively 80.71±9.0?82.01±6.88 and 67.93±8.02.Both the group A and B had a higher glucose transfer amount and activity of creatine kinase than the normal saline group,which was statistically significant(P<0.05);the difference in result between the trehalose group and fetal calf serum+10%DMSO group was not significant,and not statistically significant(P>0.05).Conclusion:The fatty tissue can still have a good activity after being preserved for 12 weeks with the 0.35mol/L trehalose solution as a cryoprotectant at-196?,indicating that,the trehalose is of a directive significance to the clinical actual application of cryoprotectant.