Effects of cancer stem cells on microtissue formation and organisation

In this thesis, it is aimed to investigate the microtissue formation ability of cancer stem cells, their localization within the microtissues and characterization techniques of these microtissues. For his purpose CD133+ cells were isolated from SaOs-2 cell line with Magnetic Cell Sorting (MACS). To evaluate microtissue formation ability, cells were cultured in different ratios in agar gels produced by 3D Petri Dish® method and observed via inverted phase contrast light microscopy. Subsequently, CD133+ cells stained with red and CD133- cells stained with green cell tracker dyes were co-cultured in agar gels in 50%, 25%, 12,5%, 10%, 5% and 2,5% ratios to investigate CD133+ cell localization in microtissues. CD133+, CD133- and SaOs-2 microtissues were characterized for CD133, Sox2, OCT3/4, Ki67 expressions with immunohistochemistry and CD133, Sox2,OCT3/4, nanog and nestin gene expression profiles with RT-PCR, and live and dead cells within microtissues were defined with Live&Dead staining method. The results show that, CD133+, CD133- and SaOs-2 cells were all able to form 3D microtissues with high cell viability, and CD133+ cells were located in microtissues randomly, without any pattern. Finally, when compared to the control group on 2D cell culture system, there was a clear difference in stemness gene expression profile in microtissues. Our results conclude that microtissues can be used as 3D tumor models to bridge in vitro culture and in vivo animal experiments for cancer research.