Experimental Study On The Effect Of Labeling Adipose Derived Stem Cells With Edu

As many study shows:human adipose tissue contains a population of cells that possesses extensive proliferative capacity and differentiates into multiple cell lineages.Of course,the easy and repeatable access to subcutaneous adipose tissue also provide a clear advantage.Based on this advantages,widely clinical implications including cell therapy and tissue engineering are highly promising.Autologous aspirated fat transfer is a popular option for soft tissue augmentation,and the top defect is the low survival rate due to partial necrosis.Study shows adipose-derived stem cells improves the efficacy of adipose transfer. Leaving the question about the exact mechanisms remain to be elucidated. ADSC persistence and niigration,and differentiation have been put forward to explain it according the studies.Yet hard evidence is needed.Our study represents the new labeling method with a new thymidine analog,5-ethynyl-2-deoxyuridine(EdU),which is replace Brdu due to many advantages.ObjectivesTo observe the cell morphology and biological characteristics of human adipose-derived stem cells with different passage time in vitro.To establish the best method and optimal concentrantion and optimal time for labeling ADSC with thymidine analog,5-ethynyl-2-deoxyuridine(EdU).To evaluate the labeled ADSC on the impact of biological characteristics,proliferation and differentiation capacity.Methods1.ADSC isolated from the expired fat tissue by digestion with type I collagenase and cultured. The primary cultured cells fusion can arrive to80%-90%in7-10days.ADSC then were purified,cultured and amplified in vitro.The configuration and morphology of the stem cells was observed under inverted microscope.ADSC at the third passage isolated and detected by flow cytometer. 2.ADSC at passage2-4were labeled with EDU by diverse concentration and diverse time.After labeled, the cell were stained by Alexa-555analyzed by fluorescence microscopy and flow cytometer.3.Labeling ADSC with5uM,24h and10uM,12h.Use the non-labeled ADSC as the blank control. The impact of EDU on the growth of ADSCs was examined by trypan blue exclusion,methylthiazoly tetrazolium(MTT) assay and assess multiple differentiation potential of ADSC using in vitro osteogenic and adipogenic induction.4.The capacity of osteogenic and adipogenic potential can be determined by Alizarin red dye and Oil Red O Staining.Finally, all the data of the research were analyzed by statistical software SPSS13.0.Results1.The adherent cells displayed themselves as fibroblast in morphology from primary culture of ADSC. From5th-7th day, the colonies grew faster and got together to form bigger colonies and get fusion in monolayer,then the cells were subcultured by trypsine.The passage ADSC proliferated fast,and could be subcultured every2-3day.The ADSC at third passage expressed surface markers of mesenchymal stem cells,including CD90,CD105,CD44,while not expressed the surface markers including CD34,CD45.2. Incorporation takes place only in replicating cells and enhances by increase incubation time and the concentration. The addition of10uM,12h and5uM,24h can both resulted in the labeling of～90%of cells and higher concentrations did not lead to increased labeling efficiency.The labeled stem cells then fixed with methanol,stained by freshly made Click-iT reaction cocktail containing Alexa-fluor555azide,cells were counterstained with Hoechst.The labeled stem cells displayed red fluorescence,with the blue fluorescence as the background.3.There was no statistically significant differences between labeled stem cells with the concentration of10uM with12h and unlabeled cells in proliferation while have statistically signigicant differences in the concentration of5uM with24h,according the MTT assay and trvpan blue exclusion.When come to the capacity of osteogenic and adipogenic potential,all data showed no statistically significant differences.ConclusionWe successfully isolate ADSC by digestion with type I coilagenase.The P3ADSC have a good biological characteristics and could been purified primarily which can be used as a powerful tool for research. Labeling with Edu in vitro can be easily performed,And labeling stem cells with the concentration of10uM with12h is the optimal concentration and time. The the optimal concentration and time of labeling has little impact on the growth of ADSC or on the differentiation and can therefore be used for ADSC trading.