Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

Autor(es)

Amaral, Robson L. F. ; Miranda, Mariza ; Marcato, Priscyla D. ; Swiech, Kamilla

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN PHYSIOLOGY; v. 8, AUG 22 2017.
• Citações Web of Science: 12

Resumo

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 x 10(4) cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 mu m) was obtained using cultures initiated with 0.5 and 1.25 x 10(4) cells/mL for the ULA method and 2.5 and 3.75 x 10(4) cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 mu g/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application. (AU)

• Processo FAPESP: 12/50762-1 - Estudo comparativo dos efeitos do imunomodulador P-MAPA encapsulado em nanopartículas poliméricas mucoadesivas versus imunoterapia com BCG na progressão do câncer de bexiga urinária: avaliação histopatológica e molecular
• Beneficiário: Priscyla Daniely Marcato Gaspari
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/05102-1 - Análise comparativa de diferentes sistemas de cultivo tridimensionais para o estabelecimento de um modelo in vitro de câncer de bexiga para testes citotoxicológicos
• Beneficiário: Robson Luis Ferraz do Amaral
• Linha de fomento: Bolsas no Brasil - Doutorado Direto