Name: Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay
Uploaded: 2023-05-19T13:30:00
Description: Watch this Scientific Journal Video about Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay at JoVE.com

microfluidic shear apparatus setup for single-cell fluid shear assay

Améliorer le diagnostic ciblé du cancer en quantifiant la mécanique unicellulaire

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.

Pleins feux sur l’auteur : Protocole d’essai de cisaillement pour la détermination des propriétés des matériaux unicellulaires  

Protocole de dosage de cisaillement pour la détermination des propriétés d’un matériau unicellulaire

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 Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay at JoVE.com

Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay  

CONFIGURATION D’UN APPAREIL DE CISAILLEMENT MICROFLUIDIQUE POUR ESSAI DE CISAILLEMENT DE FLUIDE UNICELLULAIRE

Commencez le protocole en préparant le fluide d’écoulement visqueux de l’essai de cisaillement. Pour ce faire, préchauffez le milieu de culture de base pendant environ 10 à 20 minutes à 60 à 70 degrés Celsius à l’aide d’un brassage magnétique ou d’une plaque chauffante. Tout en remuant continuellement le média, ajoutez-y doucement de la méthylcellulose pour aider les particules de méthylcellulose à se disperser rapidement sans coaguler.Laisser ce processus se poursuivre pendant environ 15 à 24 heures pour assurer un mélange homogène clair de milieu et de cellulose. Pour installer l’appareil de cisaillement, fixez un joint en caoutchouc au chemin d’écoulement pour fixer la connexion entre la chambre d’écoulement et la boîte de Pétri et assurer un flux uniforme contrôlé de cellules individuelles. Le joint en caoutchouc est disponible en différentes tailles en fonction du profil d’écoulement souhaité et de la zone d’observation.Ensuite, installez le système de test de cisaillement comprenant des seringues doubles de 60 ou 100 millilitres connectées à une pompe à seringue programmable pour la perfusion et le retrait du milieu de culture visqueux. Remplissez une seringue avec le fluide d’écoulement visqueux préparé et fixez la seringue remplie à son emplacement respectif sur la pompe à seringue. Ensuite, fixez une seringue vide à l’autre emplacement de la seringue sur la pompe à seringue.À l’aide de tubes et de connecteurs de tubes de 1/16e de pouce, connectez les deux seringues à la chambre d’écoulement. Programmez la pompe pour qu’elle perfuse et retire un certain volume de liquide à une vitesse désignée, et sélectionnez les seringues correspondantes. Un exemple de programme est affiché à l’écran.Aspirer les milieux de culture cellulaire de la boîte de Petri pour préparer le cisaillement. Ensuite, insérez et fixez la chambre d’écoulement avec le joint en caoutchouc sur la boîte de Petri contenant les cellules attachées. Placez la chambre d’écoulement microfluidique ajustée avec les cellules dans la parabole sur un microscope inversé connecté à un moniteur d’affichage.La configuration est maintenant prête à appliquer une contrainte de cisaillement fluide sur une seule cellule et à surveiller optiquement la déformation cellulaire résultante.