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Abstract(s)
A cultura bidimensional (2D) de células cancerígenas in vitro não representa fidedignamente a arquitetura tridimensional (3D), a heterogeneidade e a complexidade dos tumores humanos. Nesse sentido, são necessários modelos mais representativos, ou seja, modelos que reflitam melhor os principais aspetos da biologia tumoral. Esses são estudos essenciais de biologia e imunologia do cancro, bem como para validação de alvos e descoberta de medicamentos. Os sistemas de cultura de células tridimensionais (3D) têm vindo a ganhar um interesse crescente para a descoberta de novas drogas e engenharia de tecidos tendo em conta as suas vantagens evidentes no fornecimento de informações fisiologicamente mais relevantes e dados mais preditivos para testes in vivo. Nesta dissertação discutem-se as características dos sistemas de cultura de células 3D em comparação com a cultura 2D. Abordam-se também as principais tecnologias de cultura de células 3D e o seu impacto na descoberta de drogas, incluindo a formação de esferóides usando diferentes técnicas. Os esferóides são o resultado do crescimento e agregação de um ou mais tipos de células em cultura 3D. Em geral, os esferóides retêm as características das células iniciais, mas assumem uma forma esférica. Este trabalho consiste num processo de implementação e otimização de um protocolo para a formação de esferóides de células MDA-MB-231, que é uma linha celular de cancro de mama triplo-negativo (CMTN), através da técnica de cultura de sobreposição líquida que inclui placas com fundo em U e o uso de agarose como substrato. A resposta das células MDA-MB-231 à Doxorrubicina (DOX) foi avaliada no modelo 3D de cultura de células, e comparada com a resposta usando o modelo de cultura em monocamada (2D). Através de uma análise quantitativa baseada na área de crescimento dos esferóides, foi possível verificar que as culturas 3D mostraram maior suscetibilidade à DOX para a concentração de 4 μg/mL, na qual observou-se uma maior redução da área do esferóide. A exposição à DOX levou a uma maior diminuição do número de células viáveis no modelo de cultura 2D comparativamente com o modelo 3D, demonstrando que apresenta uma maior resistência a agentes quimioterapêuticos. Em suma, este trabalho estuda a formação, estrutura e crescimento de esferóides, bem como a sua resistência toxicológica a fármacos em comparação com a cultura de células em modelo 2D.
The in vitro two-dimensional (2D) culture of cancer cells does not represent the three-dimensional (3D) architecture, heterogeneity, and complexity of human tumors. In this sense, more representative models, i.e., that better reflect the main aspects of tumor biology. These are essential studies in cancer biology and immunology, as well as for target validation and drug discovery. Three-dimensional (3D) cell culture systems have gained increasing interest for new drug discovery and tissue engineering given their clear advantages in providing more relevant physiologically information and more predictive data for testing in vivo. This dissertation discusses the characteristics of 3D cell culture systems compared to 2D culture. The main 3D cell culture technologies and their impact on drug discovery are also discussed, including the formation of spheroids using different techniques. Spheroids are the result of the growth and aggregation of one or more cell types in 3D culture. In general, spheroids retain the characteristics of the initial cells but assume a spherical shape. This work consists of an optimization process for the formation of spheroids from MDA-MB-231 cells, which is a triple-negative breast cancer (CMTN) cell line, through the liquid overlay culture technique that includes U-bottom plates and the use of agarose as a substrate. The response of MDA-MB-231 cells to Doxorubicin (DOX) was evaluated in the 3D cell culture model and compared to a response using the monolayer (2D) culture model. Through a quantitative analysis based on the growing area of the spheroids, it was found that the 3D cultures increase the susceptibility to DOX at the concentration of 4 μg/mL, in which a greater reduction in the spheroid area was observed. Exposure to DOX led to a greater decrease in the number of viable cells in the 2D culture model compared to the 3D culture model, demonstrating that is more resistant to chemotherapeutics. In summary, this work studies the formation, structure, and growth of spheroids, as well as their toxicological drug resistance compared to cell culture in a 2D model.
The in vitro two-dimensional (2D) culture of cancer cells does not represent the three-dimensional (3D) architecture, heterogeneity, and complexity of human tumors. In this sense, more representative models, i.e., that better reflect the main aspects of tumor biology. These are essential studies in cancer biology and immunology, as well as for target validation and drug discovery. Three-dimensional (3D) cell culture systems have gained increasing interest for new drug discovery and tissue engineering given their clear advantages in providing more relevant physiologically information and more predictive data for testing in vivo. This dissertation discusses the characteristics of 3D cell culture systems compared to 2D culture. The main 3D cell culture technologies and their impact on drug discovery are also discussed, including the formation of spheroids using different techniques. Spheroids are the result of the growth and aggregation of one or more cell types in 3D culture. In general, spheroids retain the characteristics of the initial cells but assume a spherical shape. This work consists of an optimization process for the formation of spheroids from MDA-MB-231 cells, which is a triple-negative breast cancer (CMTN) cell line, through the liquid overlay culture technique that includes U-bottom plates and the use of agarose as a substrate. The response of MDA-MB-231 cells to Doxorubicin (DOX) was evaluated in the 3D cell culture model and compared to a response using the monolayer (2D) culture model. Through a quantitative analysis based on the growing area of the spheroids, it was found that the 3D cultures increase the susceptibility to DOX at the concentration of 4 μg/mL, in which a greater reduction in the spheroid area was observed. Exposure to DOX led to a greater decrease in the number of viable cells in the 2D culture model compared to the 3D culture model, demonstrating that is more resistant to chemotherapeutics. In summary, this work studies the formation, structure, and growth of spheroids, as well as their toxicological drug resistance compared to cell culture in a 2D model.
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Keywords
Cancro de mama triplo negativo Cultura 2D Cultura 3D Esferóides Doxorrubicina Triple-negative breast cancer 2D culture 3D culture Spheroids Doxorubicin