performing single-cell shear assay and imaging

Migliorare la diagnosi mirata del cancro quantificando la meccanica delle singole cellule

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.

Autore in primo piano: Protocollo di saggi a taglio per la determinazione delle proprietà dei materiali a singola cellula  

Protocollo di saggio di taglio per la determinazione delle proprietà dei materiali a singola cellula

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

Performing Single-Cell Shear Assay and Imaging  

Esecuzione di saggi e imaging a taglio a cellula singola

Per eseguire il Single-Cell Shear Assay Protocol, utilizzare la configurazione dell'apparato di taglio preassemblato costituito da una camera di flusso microfluidica montata su una piastra di Petri contenente le celle di interesse. Posizionare l'intera configurazione su un microscopio invertito con obiettivo sufficientemente alto e un monitor di visualizzazione. Focalizzare l'obiettivo del microscopio, garantendo un contrasto adeguato e bordi cellulari distinti.Spostare lo stadio del microscopio per assicurarsi che le cellule siano chiaramente visibili sul monitor del display e siano immagini dal vivo. Selezionare una cella o più celle distinte all'interno del percorso di imaging o di flusso della camera di flusso adattata e della piastra di Petri. Per mantenere un flusso di fluidi viscosi uniforme e continuo, selezionare velocità di infusione e prelievo simili.Garantire il corretto flusso laminare del fluido avente il numero di Reynold o RE inferiore a 100. Fare clic su Esegui sulla pompa di taglio per iniettare e prelevare il fluido di taglio a una velocità continua. Assicurarsi che non ci siano bolle durante l'infusione di liquidi.Iniziare a registrare un video facendo clic su Registra sul software prima che il fluido di taglio infuso entri in contatto con la cellula di interesse al microscopio. Continuare a registrare per sette minuti o per la durata desiderata dell'esposizione allo stress o fino a quando la cella di interesse taglia il fondo del piatto. Fare clic su Interrompi registrazione sul software del microscopio quando l'esecuzione è completata come desiderato.Il software salva automaticamente le immagini e i video che possono essere raccolti utilizzando le unità flash per ulteriori analisi. Estrai le immagini come file TIFF, preferibilmente a un fotogramma al secondo per una facile analisi.

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Research

JoVE Journal

Cancer Research

Performing Single-Cell Shear Assay and Imaging

123 Views. Per eseguire il Single-Cell Shear Assay Protocol, utilizzare la configurazione dell'apparato di taglio preassemblato costituito da una camera di flusso microfluidica montata su una piastra di Petri contenente le celle di interesse. Posizionare l'intera configurazione su un microscopio invertito con obiettivo sufficientemente alto e un monitor di visualizzazione. Focalizzare l'obiettivo del microscopio, garantendo un contrasto adeguato e bordi cellulari distinti.Spostare lo stadio del m...

Video: Performing Single-Cell Shear Assay and Imaging  

Shear Assay Protocol for the Determination of Single-Cell Material Properties

Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a material. A cell's resistance to stress and strain, its relaxation time, and its elasticity are all properties that can be derived and compared to other types of cells. Quantifying the mechanical properties of cancerous (malignant) versus normal (non-malignant) cells allows researchers to further uncover the biophysical fundamentals of this disease. While the mechanical properties of cancer cells are known to consistently differ from the mechanical properties of normal cells, a standard experimental procedure to deduce these properties from cells in culture is lacking. 
This paper outlines a procedure to quantify the mechanical properties of single cells in vitro using a fluid shear assay. The principle behind this assay involves applying fluid shear stress onto a single cell and optically monitoring the resulting cellular deformation over time. Cell mechanical properties are subsequently characterized using digital image correlation (DIC) analysis and fitting an appropriate viscoelastic model to the experimental data generated from the DIC analysis. Overall, the protocol outlined here aims to provide a more effective and targeted method for the diagnosis of difficult-to-treat cancers.

This protocol outlines the quantification of the mechanical properties of cancerous and non-cancerous cell lines in vitro. Conserved differences in the mechanics of cancerous and normal cells can act as a biomarker that may have implications in prognosis and diagnosis.