Melanoma is a malignant skin cancer developed from melanocytes. Treatment resistance and disease recurrence have been linked to the survival of cancer stem cells (CSCs). CSCs are highly proliferative, self-renewing cells able to differentiate into heterogeneous populations that maintain some similarities with their progenitors. This study aimed to develop three-dimensional melanosphere models containing CSCs cells that serve as a better mimic of solid tumors than other cell culture models. Melanospheres were prepared with 100 or 1000 cells/well, under non-adherent conditions using 30 µL of 2% agarose per well. Melanosphere morphology was analyzed by scanning electron microscopy (SEM) and light microscopy. For both conditions, a significant increase in melanosphere volume was observed on the 7th day compared with the 4th day of culture. Melanospheres also exhibited high cell viability and ability to form new colonies. The presence of CD271-, CD133-, and CD44-positive cells was analyzed by flow cytometry. Melanospheres showed a higher percentage of CSC markers than 2D cell cultures. Also, melanospheres cultured with lower densities showed major presence of CD44+, while higher density melanospheres showed major presence of CD271+. The findings suggest that different densities of cells are capable of enriching distinct markers of CSC in melanospheres, as an effective strategy to facilitate cancer research and therapy development.
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