The optimization of methods for the establishment of heterogeneous three-dimensional cellular models of breast cancer

BACKGROUND : Spheroids are self-assembled clusters of cells mimicking a tissue-like architecture. Since the structure of complex three-dimensional cellular models is not stable, the formation of core spheroids and further maintenance are crucial stages within the cultivation process. There are a lot of options described for the establishment of 3D cell models. A wide range of reagents is presented from simple hydrogels to complex natural and synthetic composites. However, cultivation of 3D models is still a technically challenging task requiring adaptations of protocols for particular purposes. AIM : To compare methods of the formation of gomogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and/or stromal cells of breast cancer using hydrogels such as agarose, gelatin and Matrigel™, as well as using ultra-low-adherent plates. MATERIALS AND METHODS : Breast cancer cell lines MCF7, MDA-MB-231 SK-BR-3 and stromal fibroblasts BrC4f, BrC120f, BN120f were used as a models for 3D and 3D-2 cultures. Spheroids were obtained on a substrate of simple hydrogels or when cultured on a low-adhesive plastic. The processes of formation and growth of spheroids, as well as "crushed preparations" were visualized using a Nikon Eclipse Ti-S series fluorescent inverted microscope (Nikon, Japan). RESULTS : We demonstrated, that gelatin-based hydrogel is not suitable as a substrate for obtaining 3D and 3D-2 spheroids for any of the cell lines used in the work. The use of only one type of hydrogels does not allow to obtain the entire repertoire of tumor, stromal, and heterogeneous 3D models. Agarose exhibited high output for stromal spheroids and Matrigel™ for tumor cells, and the use of ultra-low-adherent Nunclon™ Sphera™ plates was preferable for 3D-2 models combining both cell types. We also revealed that the application of cooled cultivation plastic and solutions is a technological advantage for handling spheroids growing in low-adherent plates. CONCLUSION : The proposed approaches for the formation of both homogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and (or) stromal breast cancer cells are a generalized guide to the most effective production of spheroids of various cellular composition.