Isolation, Culture And Identification Of Tumor Stem Cells Within The Ovarian Cancer OVCAR3 Cell Line In Vitro

ObjectiveTumor stem cells are a small subpopulation of cells within a tumor which have the ability to self-renew and infinite proliferation. It has been reported there exists this kind of cells with the characteristics of stem cells in many tumor tissues, which are responsible for the tumorigenesis,relapse and metastasis of the tumor. So far, the researches on the ovarial cancer stem cells are seldom concerned. In this study, the ovarial cancer stem cells were successfully obtained through the suspension culture of the ovarian cancer 0VCAR3 cell line in serum-free media. The stem cells formed spherical colonies, called "ovarispheres", and showed anchorage-independent ability. Furthermore, the bionomics and morphology of the stem cells were observed, and the expression difference of the tumor stem cells marker-ABCG2 between ovarispheres and anchorage-dependent cells was identified by using RT-PCR and Immunocytochemistry methods.MethodsThe ovarispheres were obtained and separated through the suspension culture of the ovarian 0VCAR3 cell line in serum-free medium. The morphology and growth of the tumor stem cells were observed under different culture condition including medium without B27, without using ultra-low attachment culture dish and with methylcellulose. The tumor stem cell-like characteristics was identified through the single-cell culture of the ovarian cancer 0VCAR3 cell line, the assay of tumor stem cell spheres formation and the continuous proliferation. Semi-quantitative RT-PCR,Immunocytochemistry were used to identify the expression of mRNA and protein of the tumor stem cells maker-ABCG2. Statistic analysis: the t-test was performed on the results of the Immunocytochemistry by SPSS software (version 13.0).ResultsIt was found that only a small subpopulation of cells in the ovarian cancer OVCAR3 cell line can survive in serum-free condition, and form suspended spherical colonies with the ability of continuous proliferation. B27 and ultra-low attachment culture dish have been demonstrated to be the indispensable conditions to form ovarispheres, while the suspension culture with methylcellulose can only contribute to the formation of small and irregular cell aggregates. It was further proved via single-cell culture that the ovarispheres were derived from the proliferation of single cell instead of cell aggregation. Only a few cells in the ovarispheres can form new spherical colonies, and the proportion of new colonies increased with the passage frequencies. It was also shown that the original cells of ovarispheres can self-renew and proliferate continuously. Moreover, the expression of tumor stem cells marker-ABCG2 and proteins were also higher in the ovarispheres than that in the adherent cells with serum medium.ConclusionsIn this study, it was found that there do exist a small sub-population of cells in the ovarian cancer OVCAR3 cell line that can self-renew and proliferate continuously. The cells can grow, form spherical colonies and passage in anchorage-independent,serum-free conditions. Moreover, the expression of tumor stem cells marker-ABCG2 was also higher in the ovarispheres. In a word, our studies support the theory of tumor stem cells, that is, the tumor may contain the tumor stem cells that are the origin of the tumorigenesis, relapse and metastasis of the tumor.