In vitro tumor models for cancer research and therapeutics evaluation

• Autor(a) principal: Silva, Daniel Nunes da
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/10166
• topic: Cleart; Cleart2; Esferóides; Iodeto de Propídio; Microscopia Confocal de Fluorescência; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

Spheroids have emerged as in vitro models that are able to reproduce the biological and architectural microenvironment of human tumor tissues, which make them promising platforms for cancer research purposes. Several techniques have been used in the for spheroids analysis, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), flow cytometry and fluorescence microscopy. Despite their intrinsic properties, their analysis through fluorescence microscopy, namely by confocal laser scanning microscopy (CLSM), can be very challenging due to the thickness of the spheroids as well as the light scattering phenomenon, that limits the penetration of light within the spheroids. To overcome this obstacle, several optical clearing methods have been recently explored to rend spheroids with enhanced transparency by homogenizing the refractive index (RI) of their cellular and acellular constituents, and subsequently improve the light penetration within the spheroids. Herein, it was evaluated for the first time the influence of performing the immersion of spheroids in the ClearT and ClearT2 optical clearing solutions using different incubation conditions (static, horizontal agitation and rotatory agitation), on their imaging by CLSM. Overall, the best results were obtained for those spheroids treated with both ClearT and ClearT2 under rotatory agitation. Such condition allowed the best imaging of the propidium iodide (PI) fluorescence in the Z-axis and in the spheroids’ interior. Also, ClearT demonstrated to be more suitable to perform the analysis of spheroids’ interior, while ClearT2 allowed to obtain higher PI fluorescence intensity and PI signal depth in the Z-axis. In conclusion, this work can trigger the development of new pathways for cancer therapy.