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Updated: Dec 16, 2025
Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells
Published on: June 16, 2019
Characterization of Triple-Negative Breast Cancer MDA-MB-231 Cell Spheroid Model
Zhaoming Huang1, Panpan Yu1, Jianhui Tang1
1Department of Medical Cosmetology, Xianning Central Hospital, The First Affiliated Hosptial of Hubei University of Science and Technology, Xianning, Hubei 437000, People's Republic of China.
Background:
The tumor three-dimensional (3D) spheroid model in vitro is effective on detecting malignant cells and tumorigenesis, and assessing drug resistance. Compared with two-dimensional (2D) monolayer culture, breast cancer (BC) spheroids more accurately reflect the complex microenvironment in vivo, which have been extensively reported in BC research. MDA-MB-231 cells, the triple-negative breast cancer (TNBC) cell line, display representative epithelial to mesenchymal transition (EMT) associated with BC metastasis. However, the characterization of MDA-MB-231 spheroids has been largely unknown at present, which requires further attention.
Materials And Methods:
Microwell array was conducted for the formation of MDA-MB-231 spheroids. In addition, H&E staining, immunohistochemistry (IHC), CellTiter-Glo® 3D cell viability assay, and flow cytometry were performed to investigate the structure and growth characteristics. Besides, Transwell and scratch healing assays were carried out to detect the migratory capacities compared with 2D culture. Western blotting and confocal fluorescence were selected to detect the expression of EMT-associated proteins. Additionally, the half maximal inhibitory concentration (IC50) values of antitumor compounds Carboplatin and Doxorubicin were measured to assess drug resistance.
Results:
The MDA-MB-231 spheroids were viable, which maintained a compact structure with zonation features for up to 9 days. Moreover, those spheroids had a slower growth rate than those cultured as a monolayer and differential zones of proliferation. The migratory capacities were significantly enhanced by transferring the spheroids to 2D adherent culture. Compared with 2D culture, the levels of EMT-associated proteins were significantly up-regulated in spheroids. Furthermore, toxicity assessment showed that spheroids exhibited an increased resistance to the antitumor compounds.
Conclusion:
This study develops the simple spheroids and demonstrates their structure, growth and proliferation characteristics. According to our results, the spheroids are associated with superior EMT and high resistance to toxicological response compared with the standard 2D monocultures.
