[doi: 10.5505/2017ichc.PP-49]Comparison of Some Surface Marker Expressions with Immunocytochemical and Flow Cytometric Methods in MSC Culture Passages 1 and 4Gulsemin Cicek1, Emine Utlu Ozen1, Muharrem Cicek2, Selcuk Duman1, Tahsin Murad Aktan1, Ayse Ozlem Gundeslioglu32Department of Histology and Embryology, Istanbul University, Istanbul, Turkey 3Department of Plastic and Reconstructive Surgery, Necmettin Erbakan University, Konya,Turkey INTRODUCTION: The stromal vascular fraction(SVF) sheltered in the adult human adipose tissue contains preadipocytes, mesenchymal stem cells(MSC), fibroblasts,endothelial cells, macrophages, and smooth muscle cells.Adipose tissue-derived stromal stem cells are multipotent cells that can self-renewal, proliferate and differentiate.There are environmental conditions of the cells that provide and stabilize these essential features of the MSCs.Repair mechanisms in cellular treatments are achieved by these conditions.There are clusters of differentiation(CD) markers on the surface of there cells, which play a role in adhesion and migration or affect cell signaling pathways.MSCs express primarily CD44, CD73, CD90, CD105, CD166 on their surface while; HLA-DR, CD19 and CD34 are not expressed.The aim of this study is to evaluate surface marker expressions in the progressive culture passages of MSC.METHODS: SVF cells were harvested with enyzimatic reaction of human adipose tissue obtained from liposuction procedure.Cell cultivation was done with 25 cm² flasks by using DMEM with additives of 10% fetal bovine serum, penicillin-streptomycin and L-glutamin.When cellular confluence in culture flask reached 70-80%; the cells were detached and replated onto sterile culture flasks of to 25 cm² as 1000 cells per cm².Immunocytochemical stain and flow cytometric analysis was performed on P1 and P4 passages cells.The expression of CD19, CD44, CD90, CD105 in MSC was determined by immunocytochemistry using primary polyclonal antibodies (Bioss,USA) at a dilution of 1:150 with secondary avidin-biotin-peroxidase complex (Ultravision Detection System Anti-polyvalent,HRP,LabVision,USA).For flow cytometric analysis the cells were detached from the flask bottom and incubated in the dark for 30 min at +4°C temperature after being treated with conjugated antibodies anti-human CD105/FITC, anti-human CD90/PE, anti-human CD44/Alexa Fluor® 647 and anti-human CD19/PECy5 (MerckMillipore,Germany).RESULTS: Flow cytometric analysis showed that surface marker expressions are as; for P1:CD19 0,6%; CD44 96,2%; CD105 79,3%; CD90 97,7% and for P4:CD19 0,6%; CD44 93,4%; CD105 78,8%; CD90 96,8% positive in rates while immunocytochemical staining results also supported these results.In conclusion, we observed that between p1 and p4 passages there is not any significant difference in studied MSC surface marker expressions.Ethics approval:This study was conducted with the approval of the ethics committee of Meram Medical Faculty, Necmettin Erbakan University(Ref:2016/674) |