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

シングルセルメカニクスの定量化によるがん標的診断の強化

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.

著者スポットライト:シングルセル材料特性の決定のためのせん断アッセイプロトコル  

単一セル材料特性を決定するためのせん断アッセイプロトコル

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  

シングルセルせん断アッセイとイメージングの実行

シングルセルせん断アッセイプロトコルを実行するには、目的の細胞を含むペトリ皿に取り付けられたマイクロ流体フローチャンバーで構成される組み立て済みのせん断装置セットアップを使用します。セットアップ全体を、十分に高い対物レンズとディスプレイモニターを備えた倒立顕微鏡に置きます。顕微鏡対物レンズの焦点を合わせ、適切なコントラストと明瞭な細胞エッジを確保します。顕微鏡ステージを動かして、細胞がディスプレイモニターにはっきりと見え、ライブ画像であることを確認します。適合フローチャンバーとペトリ皿のイメージングまたは流路内の1つのセルまたは複数の異なるセルを選択します。連続的に均一な粘性媒体の流れを維持するには、同様の注入速度と離脱速度を選択します。レイノルド数またはREが100未満の流体の適切な層流を確保します。せん断ポンプの[実行]をクリックして、せん断液を連続速度で注入および回収します。液体の注入中に気泡がないことを確認してください。注入されたせん断液が顕微鏡下で目的の細胞と接触する前に、ソフトウェアの[録画]をクリックしてビデオの録画を開始します。7分間、またはストレス曝露の希望の期間、または目的の細胞が皿の底から剪断されるまで記録を続けます。顕微鏡ソフトウェアの[記録の停止]をクリックします 必要に応じて実行が完了します。ソフトウェアは自動的にさらなる分析のためにフラッシュドライブを使用して収集することができる画像やビデオを保存します。画像をTIFFファイルとして抽出し、できれば1秒あたり1フレームで抽出して、簡単に分析できるようにします。

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