An efficient and practical method for culturing human ovarian surface epithelial cells / 中国组织工程研究

ABSTRACT

BACKGROUND: It is difficult to in vitro isolate and culture the ovarian surface epithelium with high purity, strong vitality and stable biological characteristics. Tissue adherence and enzymatic digestion are commonly used for primary culture, but there are certain problems in cel col ection, cel viability and cel purity. OBJECTIVE: To establish a method for primary isolating, culturing and identifying human ovarian epithelium. METHODS: The ovarian surface epithelium was obtained with cel brush innovatively, and then the cells were isolated and purified with erythrocyte spal ation and differential adherence. The epidermal growth factor was added into the serum-free Dulbecco’s modified Eagle’s medium-F12 medium for cel culture. The cel morphology was observed under inverted microscope, and hematoxylin-eosin staining and immunocytochemical staining were used to identify the cells, then the growth curve was draw. RESULTS AND CONCLUSION: The ovarian surface epithelium became adherent after cultured for 24 hours, and reached fusion basical y after cultured for 6-12 days. The cells were polygonal or flat with strong transparency and refraction. The morphological characteristics of the cells were in line with those of the normal epithelial cells, and almost al the isolated cells could express the epithelial cells surface marker CK19. The cells could be passaged for 6-8 generations with wel growth and the cel growth curve was in “S” shape. The purity of the cells was more than 95%. The results suggest that cel brush method is simple to operate and can obtain a large amount of ovarian surface epithelium rapidly. The purity of the isolated cells can reach to 95% after treated with erythrocyte spal ation and differential adherence method and the cells are in stable growth.