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Publication
The use of 3D cellular models for genotoxicity studies.
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Abstract(s)
O desenvolvimento de ensaios de genotoxicidade envolve várias etapas, tendo inicio nas
metodologias in vitro, seguido por in vivo, até ser passível a translação para seres humanos. O
principal objetivo deste projeto foi comparar a resposta genotóxica das linhas celulares 2D
(bidimensionais) com os modelos celulares tridimensionais (3D). Para ambos os modelos,
utilizaram-se as mesmas linhas celulares de mama (tumorais e não tumorais: MCF-7 e MCF-
10A).
A implementação de esferoides 3D foi realizada com sucesso utilizando as linhas
celulares de mama MCF-7 e MCF-10A. O próximo passo foi o desenvolvimento de ensaios de
genotoxicidade em modelos esferoidais. Neste projeto, recorreu-se ao Comet Assay, também
conhecido como ensaio de eletroforese em gel de célula única. De forma a validar este ensaio
em modelos 3D, foram realizadas experiências com vista a analisar e comparar as diferenças
nos resultados obtidos para os modelos de cultura 2D e 3D. O Comet Assay permitiu avaliar a
resposta celular da exposição a agentes químicos indutores de lesões no DNA, sendo que o
tamanho da cauda pode ser diretamente correlacionado a uma maior lesão no DNA celular. Para
a induções de lesões no DNA, recorreu-se ao uso da doxorrubicina (DOX), um agente
quimioterapêutico utilizado no tratamento do cancro. O controlo positivo utilizado neste estudo
foi o peróxido de hidrogénio (H2O2).
Os resultados revelaram que, para ambas as linhas celulares e ambos os agentes (DOX
e H2O2), os modelos 2D exibem níveis mais altos de lesão no DNA em comparação com seus
equivalentes em 3D. Esta disparidade é particularmente pronunciada na linha celular MCF-
10A. No entanto, ensaios adicionais e complementares devem ser realizados, sendo
indispensáveis para avaliar a reprodutibilidade e a significância estatística dos resultados
obtidos. Com este estudo concluiu-se que a validação do uso do Comet Assay para modelos
esferoidais 3D necessita ser mais profundamente estudada, incluindo a caracterização
fenotípica e morfológica dos esferoides.
The development of genotoxicity assays involves various stages, progressing in the complexity of models utilized in studies, starting from in vitro, followed by in vivo, until extrapolation to humans is achieved. The main goal of this project was comparing the genotoxic response for assessment through functional assays using 2D cell cultures (monolayers) and implementing of 3D spheroidal cultures from the same breast cell lines (tumoral and non- tumoral: MCF-7 and MCF-10A). The implementation of 3D spheroid was successfully achieved using MCF-7 and MCF- 10A breast cell lines. The next step was the development of genotoxicity assays to be implemented in spheroid models. The assay selected to evaluate genotoxicity values was the Single Cell Gel Electrophoresis, commonly known as Comet Assay. To validate this assay, we also carried-out experiments using this methodology in 2D cell lines (MCF-7 and MCF-10A). The Comet Assay allows for an evaluation of cellular response through exposure to chemical agents inducing DNA damage, where the tail size (DNA breaks) is directly correlated with a higher cell DNA damage. The chemical agents used to induce the DNA damage were doxorubicin (DOX), a chemotherapeutic agent used in cancer treatment, and hydrogen peroxide (H2O2) as positive control. The main objective of this study was to determine if it is possible to extrapolate from well-established 2D assays to more complex models like 3D spheroids, possibly yielding results biologically more realistic that provide reliable insights into cellular mechanisms. Our results revealed that for both cell lines and both agents (DOX and H2O2), the 2D models exhibit higher levels of DNA damage in comparison to their 3D counterparts. This disparity is particularly pronounced in the MCF-10A model types. Nevertheless, further assays should be performed which will be indispensable to rigorously evaluate the reproducibility and significance of the observed trends. The validation of 3D spheroid models needs to be more deeply studied, including phenotypical and morphological characterization of spheroids.
The development of genotoxicity assays involves various stages, progressing in the complexity of models utilized in studies, starting from in vitro, followed by in vivo, until extrapolation to humans is achieved. The main goal of this project was comparing the genotoxic response for assessment through functional assays using 2D cell cultures (monolayers) and implementing of 3D spheroidal cultures from the same breast cell lines (tumoral and non- tumoral: MCF-7 and MCF-10A). The implementation of 3D spheroid was successfully achieved using MCF-7 and MCF- 10A breast cell lines. The next step was the development of genotoxicity assays to be implemented in spheroid models. The assay selected to evaluate genotoxicity values was the Single Cell Gel Electrophoresis, commonly known as Comet Assay. To validate this assay, we also carried-out experiments using this methodology in 2D cell lines (MCF-7 and MCF-10A). The Comet Assay allows for an evaluation of cellular response through exposure to chemical agents inducing DNA damage, where the tail size (DNA breaks) is directly correlated with a higher cell DNA damage. The chemical agents used to induce the DNA damage were doxorubicin (DOX), a chemotherapeutic agent used in cancer treatment, and hydrogen peroxide (H2O2) as positive control. The main objective of this study was to determine if it is possible to extrapolate from well-established 2D assays to more complex models like 3D spheroids, possibly yielding results biologically more realistic that provide reliable insights into cellular mechanisms. Our results revealed that for both cell lines and both agents (DOX and H2O2), the 2D models exhibit higher levels of DNA damage in comparison to their 3D counterparts. This disparity is particularly pronounced in the MCF-10A model types. Nevertheless, further assays should be performed which will be indispensable to rigorously evaluate the reproducibility and significance of the observed trends. The validation of 3D spheroid models needs to be more deeply studied, including phenotypical and morphological characterization of spheroids.
Description
Keywords
Cultura Celular 2D Cultura Celular 3D Genotoxicidade Comet Assay Esferóides 2D Cell Culture 3D Cell Culture Genotoxicity Spheroids