Morphometrical, Morphological, and Immunocytochemical Characterization of a Tool for Cytotoxicity Research: 3D Cultures of Breast Cell Lines Grown in Ultra-Low Attachment Plates

Abstract

Three-dimensional cell cultures may better mimic avascular tumors. Yet, they still lack characterization and standardization. Therefore, this study aimed to (a) generate multicellular aggregates (MCAs) of four breast cell lines: MCF7, MDA-MB-231, and SKBR3 (tumoral) and MCF12A (non-tumoral) using ultra-low attachment (ULA) plates, (b) detail the methodology used for their formation and analysis, providing technical tips, and (c) characterize the MCAs using morphometry, qualitative cytology (at light and electron microscopy), and quantitative immunocytochemistry (ICC) analysis. Each cell line generated uniform MCAs with structural differences among cell lines: MCF7 and MDA-MB-231 MCAs showed an ellipsoid/discoid shape and compact structure, while MCF12A and SKBR3 MCAs were loose, more flattened, and presented bigger areas. MCF7 MCAs revealed glandular breast differentiation features. ICC showed a random distribution of the proliferating and apoptotic cells throughout the MCAs, not fitting in the traditional spheroid model. ICC for cytokeratin, vimentin, and E-cadherin showed different results according to the cell lines. Estrogen (ER) and progesterone (PR) receptors were positive only in MCF7 and human epidermal growth factor receptor 2 (HER-2) in SKBR3. The presented characterization of the MCAs in non-exposed conditions provided a good baseline to evaluate the cytotoxic effects of potential anticancer compounds.

Effects and Mechanisms of Action of Preussin, a Marine Fungal Metabolite, against the Triple-Negative Breast Cancer Cell Line, MDA-MB-231, in 2D and 3D Cultures

“…Therefore, the MDA-MB-231 MCAs architecture and the cell cohesiveness may reduce preussin and Dox penetration, resulting in a lower bioavailability of the compounds to all cells. It is important to note, however, that different authors have described the obtention of MDA-MB-231 cell line 3D structures with varying levels of cohesiveness [ 58 , 60 , 64 , 65 , 66 , 67 , 68 , 69 ] and varying resistance to drugs (such as Dox) when we compared 2D with 3D cell cultures [ 60 , 64 ]. Malhão et al [ 65 ], using the same cell line and method to obtain the 3D culture, have found the MCAs of MDA-MB-231 with an ellipsoid form while maintaining cohesive 3D structures.…”

“…It is important to note, however, that different authors have described the obtention of MDA-MB-231 cell line 3D structures with varying levels of cohesiveness [ 58 , 60 , 64 , 65 , 66 , 67 , 68 , 69 ] and varying resistance to drugs (such as Dox) when we compared 2D with 3D cell cultures [ 60 , 64 ]. Malhão et al [ 65 ], using the same cell line and method to obtain the 3D culture, have found the MCAs of MDA-MB-231 with an ellipsoid form while maintaining cohesive 3D structures. In this study, the existence of cell adhesions, as observed in the ultrastructural analysis of these MCAs, supports the previous findings [ 65 ] and further confirms the cohesiveness of the MDA-MB-231 MCAs.…”

“…Malhão et al [ 65 ], using the same cell line and method to obtain the 3D culture, have found the MCAs of MDA-MB-231 with an ellipsoid form while maintaining cohesive 3D structures. In this study, the existence of cell adhesions, as observed in the ultrastructural analysis of these MCAs, supports the previous findings [ 65 ] and further confirms the cohesiveness of the MDA-MB-231 MCAs.…”

“…The presence of the necro-apoptotic core in the 3D cell culture is in agreement with that described in the literature, mainly in the classically described spheroids [ 45 , 97 ]. Even in the new and more complex model of cell distribution within the MCAs proposed by Malhão et al [ 65 ], it is still possible to observe a higher number of cells in the processes of apoptosis and necrosis, mainly in the center of the MCAs.…”

3D co-culture of macrophages and fibroblasts in a sessile drop chip for unveiling the role of macrophages in skin wound-healing

“…They include microfluidic devices, polymeric hard scaffolds, hydrogel scaffolds, microplates, micropatterned surfaces, and spheroids. Scaffold-free techniques rely on the self-aggregation of cells in specialized culture plates, such as hanging drop microplates 23 , low adhesion plates with ultra-low attachment coating that promotes the spheroid formation 24, 25 , and micropatterned plates that allow for microfluidic cell culture 26 . The development of spheroids is the most popular and convenient technique for 3D cell co-culture.…”