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

通过量化单细胞力学增强靶向癌症诊断

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.

作者聚焦：用于测定单细胞材料特性的剪切测定实验方案  

用于测定单细胞材料特性的剪切测定方案

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  

用于单细胞流体剪切测定的微流体剪切装置设置

通过制备剪切测定粘性流动介质开始协议。为此，使用磁力搅拌或热板将基础培养基在60至70摄氏度下预热约10至20分钟。在不断搅拌介质的同时，轻轻地将甲基纤维素添加到其中，以帮助甲基纤维素颗粒快速分散而不会凝结。让此过程持续约15至24小时，以确保培养基和纤维素的清晰均匀混合物。要设置剪切装置，请将橡胶垫圈连接到流路以固定流动室和培养皿之间的连接，并确保单个细胞的受控均匀流动。橡胶垫圈有不同的尺寸，具体取决于所需的流量曲线和观察区域。接下来，设置剪切测定系统，该系统由连接到可编程注射泵的60或100毫升双注射器组成，用于输注和取出粘性培养基。用准备好的粘性流动介质填充注射器，并将填充的注射器连接到其在注射泵上的相应位置。然后，将空注射器连接到注射泵上的另一个注射器位置。使用 1/16 英寸管道和管道连接器，将两个注射器连接到流动室。对泵进行编程，以指定的速率注入和抽出一定体积的液体，并选择相应的注射器。屏幕上显示了一个示例程序。从培养皿中吸出细胞培养基以准备剪切。接下来，将带有橡胶垫圈的流动室插入并固定到包含附着细胞的培养皿上。将装有培养皿中的细胞的安装的微流体流动室放在连接到显示器的倒置显微镜上。该装置现在已准备好将流体剪切应力施加到单个电池上，并光学监测由此产生的细胞变形。