| ObjectiveExplore the adipose-derived stem cells (Adipose-derived stem/stromalcells, ASCs) diameter, surface phenotype and differentiation of biologicalcharacteristics.Methods1.Cell culture: Adipose tissue were derived from the department of plasticsurgery, General Hospital of Shenyang Military Region5liposuction patients.Adipose tissue were digested with0.1%Ⅰtype for45mins under asepticconditions. Then the density gradient centrifugation was used to get stromalvascular component (stromal vascular fraction, SVF) including ASCs.The cellswere seeded and cultured with25cm2culture bottle under37°C and5%CO2culture condition. The medium was changed after48h to get rid of unadherentcells. The medium was changed every3days and the cell were passaged aftergeting80%~90%confluence.2.Cell diameter test: The Cellometer cell counting syetem were used fordetection the diameter of cells form primary passage to the third generation (P0to P3) cell diameter. Every time detection repeated for five times, and the datawere showed by average±standard deviation.T-test were used for Statisticalanalysis.3.Cell phenotypes detection: The third passage cells were used fordectection. Trypsin digestion the single cells`suspensions, were added withCD31, CD45, CD49d,CD34,CD105and CD106antibody20μl, respectively.Thesuspensions without antibodies were as control group. The suspension wereincubated for15mins without light and centrifugated for15mins under200×g.PBS wash and centrifugated for3mins under200×g.Flow cytometry was used for cell phenotype analysis.4. Differentiation into adipocytes: P3generation cells were cultured withadipogenic induction medium (DMEM,10%FBS,1μmol/L dexamethasone,200umol/L indomethacin,0.5μmol/L3-isobutyl-1-methyl xanthine,10μg/ml insulin.Filtering).After inducing for7days, oil red O stain were carried out under roomtemperature for15mins.The results were observed10images were collectedby random. In view of all cells were calculated and adipogenic cell number,calculate the adipogenic induction rate. The induced rate were calculated withthe formulation of adipogenic induction rate equals to all adipogenic cell number/total cell number x100%.Results1.The configuraton of cells:SVF cells adherenced after seed in25cm2culture flasks for24to48h. Cells`configuration varied by spindle, polygonal(including triangular and square cells mostly) and oval shape. Subsequently,the spindle cells and polygonal cells gradually dominatly, covered the bottom ofthe bottle10days. After passage, cells grow faster and long spindle-shapedcells dominated.2.The diameter of cells:The average diameter of the unadherent cells were9.40±1.89μm, in which76.15%cell diameter was7.45~9μm, with the minimumdiameter of7.45μm cells, the largest cell diameter was29.18μm.The primarypassage of cell average diameter was13.26±1.64μm, of which60.81%celldiameter of7.42~15μm. With the minimum diameter of7.42um cells and thelargest cell diameter of28.66μm.The second generation cells average diameterof13.66±0.97μm, of which91.67%cell diameter range of7.22~19.07μm. Withthe minimum cell diameter of7.22μm and the largest cell diameter of25.65μm.The third generation cells for15.44±0.97μm, of which76.92%cell diameterrange of7.47~18.49μm. With the minimum diameter of7.47μm cells and thelargest cell diameter of29.50μm.3.Cell phenotype detects:Flow cytometric analysis show that the cellsurface phenotyp was CD31-CD34-CD45-CD49d+CD105+CD106-.CD31was0.67±0.56%,CD34was2.45±1.68%, CD45was1.19±0.61%, CD105was 32.33±33.79%. CD49d was31.94±14.37%, CD106was1.41±0.73%.4. Differentiation into adipocytes: Differention into adipocytes for48~72h,the cells morphology change gradually. The long spindle cells sharp becomethe polygons or circular cells.The "rooming" shape of small lipid drops appearedin the cytoplasm, initially located in the nucleus outside. The number of lipiddrops and volume increased by the induction time.The oil stain demonstratesthe exist of the adipocytes. At the same time, the rounded cells give priority tothe long spindle cells rare.After random counting,the fat induction rate is51.12±8.01%.ConclusionThis study isolated human adipose-derived stem cells and culture them invitro.After24h inoculation the circular clear cell change to long fusiform cells.Using Cellometer cell count system, the cell size differentiated and change withthe culture time.Flow system cytometry confirmed, the passage third cellsphenotypes was CD31-CD34-CD45-CD49d+CD105+CD106-.In vitro cells candifferentiation into adipocytes under adipogenic conditions... |
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