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Non-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly. In this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated. Our results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres. The described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres.
Gao et al. (2018) studied this question.