Name: Performing Single-Cell Shear Assay and Imaging
Uploaded: 2023-05-19T13:30:00
Description: Watch this Scientific Journal Video about Performing Single-Cell Shear Assay and Imaging at JoVE.com

performing single-cell shear assay and imaging

Aprimorando o diagnóstico de câncer direcionado quantificando a mecânica de uma única célula

Studying the biophysical differences between cancerous and non-cancerous cells allows for novel diagnostic and therapeutic opportunities1. Understanding how differences in biomechanics/mechanobiology contribute to tumor progression and treatment resistance will reveal new avenues for targeted therapy and early diagnosis2.
While it is known that cancer cell mechanical properties differ from normal cells (e.g., viscoelasticity of the plasma membrane and nuclear envelope)3,4,5, robust and reproducible methods for measuring these properties in live cells are lacking6. The shear assay method is used to quantify the mechanical properties of cells by subjecting single cells to fluid shear stress and analyzing their individual responses and resistance to the applied stress3,4,5,7,8,9. Although several methods and techniques have been used to characterize the mechanical properties of single cells, these tend to affect cell material properties by i) perforating/damaging the cell membrane due to the indentation depth, complex tip geometries, or substrate stiffening associated with atomic force microscopy (AFM)10,11, ii) inducing cellular photodamage during optical trapping12,13, or iii) inducing complex stress states associated with micropipette aspiration14,15. These external effects are associated with significant uncertainties in the accuracy of cell viscoelasticity measurements6,16,17.
To address these limitations, the shear assay method described here provides a highly controllable and simple approach to simulate physiological flow in the body without affecting cellular material properties in the process. Fluid shear stresses in this assay represent mechanical stresses experienced by cells in the body either by fluids within the tumor interstitium or in the blood during circulation18,19,20. Further, these fluid stresses promote various malignant behaviors in cancer cells, including progression, migration, metastasis, and cell death19,21,22,23&#160;which vary between tumorigenic and non-tumorigenic cells. Moreover, the altered mechanical features of cancer cells (i.e., they are often &#34;softer&#34; than normal cells found within the same organ) allow them to persist in hostile tumor microenvironments, invade surrounding normal tissues, and metastasize to distant sites24,25,26. By creating a pseudo-biological environment where cells experience physiological levels of fluid shear stress, a process that is physiologically relevant and not destructive to the cell is achieved. The cellular responses to these applied fluid shear stresses allow us to characterize cell mechanical properties.
This paper provides a shear assay protocol for the extensive study of the mechanical properties and behavior of cancerous and non-cancerous cells under applied shear stress. Cells respond to external forces in an elastic and viscous manner and can therefore be idealized as a viscoelastic material3. This technique is categorized into: (i) cell culture of dispersed single cells, (ii) controlled application of fluid shear stress, (iii) in situ imaging and observation of cellular behavior (including resistance to stress and deformation), (iv) strain analysis of cells to determine the extent of deformation, and (v) characterization of the viscoelastic properties of single cells. By interrogating these mechanical properties and behaviors, complex cellular mechanobiology can be distilled to quantifiable data. A protocol outlining this method allows for the cataloging of and comparison between various malignant and non-malignant cell types. Quantifying these differences has the potential to establish diagnostic and therapeutic biomarkers.

Autor em destaque: Protocolo de ensaio de cisalhamento para a determinação de propriedades de material de célula única  

Protocolo de ensaio de cisalhamento para a determinação de propriedades de material de célula única

The physical hallmarks of cancer are relatively new and exciting topics in cancer research. Our research seeks to specifically characterize the mechanical properties of both cancers and non-cancer cells to better understand the physical mechanisms by which cancer cells survive shear forces in the body and invade the immune system. Recent development in my period of research is the use of microfluidic devices and high-throughput techniques to understudy in a larger scale, cellular deformability and the viscoelastic properties of cells, and also in the identification of novel biomarkers such as the regulation of cytoskeletal proteins and extracellular matrix proteins for early cancer diagnosis.Current experimental challenges involved in this field are the complexity of the tumor microenvironment, scaling throughput, clinical translation, standardization and reproducibility, and integration of multimodal data. So, some of the significant findings I've established in this field from my past research and from my current research is that cancer cells can be distinguished from normal cells based on their responses to mechanical stressors. And the extent to which resist mechanical stressors is dependent on structural integrity, which is dictated to a reasonable extent by the presence of cytoskeletal proteins like the filamentous actin and other structural proteins within the cell.Our protocol offers a simple, repeatable, and non-destructive approach to characterize cultured cells mechanically. This is in contrast to other techniques like AFM and optical tweezers, which require a certain level of technical expertise in order to perform an experiment in a relatively low throughput. Within the use of the shear acid technique, will greatly advance the single cell mechanical characterization, and will also aid in many different cross-sectional areas such as rare disease diagnosis, personalized diagnosis and care, and monitoring single-cell drug interactions.

Watch this Scientific Journal Video about Performing Single-Cell Shear Assay and Imaging at JoVE.com

Performing Single-Cell Shear Assay and Imaging  

Executando ensaio de cisalhamento de célula única e geração de imagens

Para realizar o Protocolo de Ensaio de Cisalhamento de Célula Única, utilize o arranjo de aparelho de cisalhamento pré-montado que consiste em uma câmara de fluxo microfluídica encaixada colocada em uma placa de Petri contendo as células de interesse. Coloque toda a configuração em um microscópio invertido com objetiva alta o suficiente e um monitor de exibição. Focalizar a objetiva do microscópio, garantindo contraste adequado e bordas celulares distintas.Mova o estágio do microscópio para garantir que as células sejam claramente visíveis no monitor de exibição e sejam imagens ao vivo. Selecione uma célula ou várias células distintas dentro da imagem ou do caminho de fluxo da câmara de fluxo ajustada e da placa de Petri. Para manter um fluxo contínuo e uniforme de meios viscosos, selecione taxas de infusão e retirada semelhantes.Garantir o fluxo laminar adequado do fluido com o número de Reynold ou RE inferior a 100. Clique em Executar na bomba de cisalhamento para injetar e retirar o fluido de cisalhamento a uma taxa contínua. Certifique-se de que não há bolhas durante a infusão de fluidos.Comece a gravar um vídeo clicando em Gravar no software antes que o fluido de cisalhamento infundido entre em contato com a célula de interesse sob o microscópio. Continue a gravar por sete minutos ou pela duração desejada da exposição ao estresse ou até que a célula de interesse se desfaça do fundo do prato. Clique em Parar gravação no software do microscópio quando a execução for concluída conforme desejado.O software salva automaticamente as imagens e vídeos que podem ser coletados usando unidades flash para análise posterior. Extraia as imagens como arquivos TIFF, de preferência a um quadro por segundo para facilitar a análise.

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