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    Estabelecimento e caracterização de cultura primária de tecido vaginal. Viabilidade do uso de terapia celular no tratamento de pacientes com síndrome de Mayer-Rokitansky-Küster-Hauser
    (Universidade Federal de São Paulo (UNIFESP), 2015-12-31) Paula, Tatiane Aparecida de [UNIFESP]; Girão, Manoel Joao Batista Castello [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The uterine and vaginal agenesis, also known as Syndrome Mayer-Rokitansky-Küster-Hauser (SMRKH) is a congenital malformation that, although rare, carries important clinical and psychological repercussions, due to the impairment of sexual activity and reproductive life. The treatment for this disease is divided into bloodless and surgical methods and consists in the construction of a neovagina, allowing the patient to have a normal sexual life. However, there is no consensus on best approach of treatment because of the advantages and disadvantages presented in both cases. Recently experimental studies described a successful technique of rabbit vaginal tissue engineering using samples of vagina and muscle autologous cells. In 2007 it was reported the first neovaginoplasty case with the MacIndoe modified technique were it was used the patient's own cells to produce an autologous vaginal tissue in the laboratory. Objectives: Our aim is to use the patient's own cells to produce an autologous vaginal tissue in the laboratory to be used in the near future in neovaginoplasty. Methods: We performed 05 full-thickness biopsies from vaginal vestibule of ~ 1cm2. After several washes in PBS with antibiotics, the tissue was minced into small pieces which were then put in culture plates with DMEM/F-12 supplemented with 10% of fetal bovine serum to allow cells to migrate from the explants. After cells reached 70% of confluence, the explants were removed so cells could grown to remaining spaces, at this moment the plates were harvested and cells were frozen in this first passage at density of 106 cells/vial, if we could not obtain this cell density, cells were further expanded and frozen at second passage. Using flow citometry, 03 samples were characterized using the following antibodies: CD90, CD73, CD105 and STRO-I for mesenchymal stem cells as well as pan-citokeratyn, CD133, CD9, CD227 and CD1d for epithelial cells, being the last two specifically used for vaginal epithelial cells. Finally we used CD34 for hematopoietic progenitor cells and vimentin for fibroblasts. The data was acquired using FACS Canto II ?BD Bioscience and analyzed with FlowJo 7.6.5 software. Results: Using explant technique we were able to obtain a mixed cell population of viable cells. These cells were characterized by 67% of mesenchymal stem cells (CD90+ CD73+ CD105+; SD=10,18,); we find high positivity of STRO-I cells (75,2%, SD=8,77) and vimetin (80,3%, SD=4,59). For most epithelial markers used we also find high values: 71,9% of CD133, 73,7% of CD9 and 80,3% of CD227 (SD= 6,7, 5,74 and 8,77 respectively). We found a weak positivity of citokeratyn and PDGFR-RB cells among the samples (13,6%, 0,52%, and 1,96%; 0,93%, 0,38%, 0,20% respectively) and a weak positivity of CD34 cells (14,7%, SD= 4,98). Conclusions: Using explant technique we were able to establish primary cell cultures from vaginal biopsies. Those cultures are characterized by having a mixed population of cells, namely, stem cells, epithelial cells and fibroblasts, showing that this approach could be used to obtain autologous vaginal tissue.