performing single-cell shear assay and imaging

שיפור אבחון סרטן ממוקד על ידי כימות מכניקה של תא בודד

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.

מחבר זרקור: פרוטוקול Shear Assay לקביעת תכונות חומר חד-תאי  

פרוטוקול בדיקת גזירה לקביעת תכונות חומר חד-תאי

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  

ביצוע בדיקת גזירה והדמיה של תא בודד

כדי לבצע את פרוטוקול בדיקת הגזירה של תא יחיד, השתמש במערך מנגנון הגזירה שהורכב מראש המורכב מתא זרימה מיקרופלואידי מותאם המונח על צלחת פטרי המכילה את התאים המעניינים. מקם את כל המערך על מיקרוסקופ הפוך עם מטרה גבוהה מספיק וצג תצוגה. מקדו את מטרת המיקרוסקופ, תוך הבטחת ניגודיות מספקת וקצוות תאים ברורים.הזז את שלב המיקרוסקופ כדי לוודא שהתאים נראים בבירור על צג התצוגה והם תמונות חיות. בחר תא אחד או תאים נפרדים מרובים בתוך נתיב ההדמיה או הזרימה של תא הזרימה המותאם וצלחת הפטרי. כדי לשמור על זרימת מדיה צמיגה אחידה רציפה, בחר שיעורי עירוי ונסיגה דומים.ודא זרימה למינרית תקינה של הנוזל עם מספר ריינולד או RE פחות מ -100. לחץ על הפעל במשאבת הגזירה כדי להזריק ולהוציא את נוזל הגזירה בקצב רציף. ודא שאין בועות במהלך עירוי נוזלים.התחל להקליט וידאו על ידי לחיצה על הקלט בתוכנה לפני שנוזל הגזירה המוחדר יוצר מגע עם התא המעניין מתחת למיקרוסקופ. המשיכו להקליט במשך שבע דקות או למשך הזמן הרצוי של החשיפה לסטרס או עד שהתא המעניין חותך את תחתית המנה. לחץ על הפסק הקלטה בתוכנת המיקרוסקופ כאשר הריצה תושלם כרצונך.התוכנה שומרת באופן אוטומטי את התמונות והסרטונים אשר ניתן לאסוף באמצעות כונני הבזק לניתוח נוסף. חלץ את התמונות כקובצי TIFF, רצוי במסגרת אחת לשנייה לצורך ניתוח פנים.

performing-single-cell-shear-assay-and-imaging

Research

JoVE Journal

Cancer Research

Performing Single-Cell Shear Assay and Imaging

118 Views.  University of Notre Dame. כדי לבצע את פרוטוקול בדיקת הגזירה של תא יחיד, השתמש במערך מנגנון הגזירה שהורכב מראש המורכב מתא זרימה מיקרופלואידי מותאם המונח על צלחת פטרי המכילה את התאים המעניינים. מקם את כל המערך על מיקרוסקופ הפוך עם מטרה גבוהה מספיק וצג תצוגה. מקדו את מטרת המיקרוסקופ, תוך הבטחת ניגודיות...

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.

Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a ...

Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay

Begin the protocol by preparing the shear assay viscous flow media. To do so, preheat the base culture media for about 10 to 20 minutes at 60 to 70 degrees Celsius using a magnetic stir or hot plate. While continuously stirring the media, gently add methylcellulose into it to help the methylcellulose particles disperse quickly without coagulating.Allow this process to continue for approximately 15 to 24 hours to ensure a clear homogenous mix of media and cellulose. To set up the shear apparatus, attach a rubber gasket to the flow path to fasten the connection between the flow chamber and the Petri dish, and ensure a controlled uniform flow of single cells. The rubber gasket comes in different sizes depending on the desired flow profile and area of observation.Next, set up the shear assay system comprising of 60 or 100 milliliter dual syringes connected to a programmable syringe pump for infusion and withdrawal of the viscous culture media. Fill a syringe with the prepared viscous flow media and attach the filled syringe to its respective location on the syringe pump. Then, attach an empty syringe to the other syringe location on the syringe pump.Using 1/16th inch tubing and tubing connectors, connect both syringes to the flow chamber. Program the pump to infuse and withdraw a certain volume of fluid at a designated rate, and select the corresponding syringes. An example program is shown on the screen.Aspirate the cell culture media from the Petri dish to prepare for shear. Next, insert and fix the flow chamber with the rubber gasket onto the Petri dish containing the attached cells. Place the fitted microfluidic flow chamber with the cells in the dish onto an inverted microscope connected to a display monitor.The setup is now ready for applying fluid shear stress onto a single cell and optically monitoring the resulting cellular deformation.

הגדרת מנגנון גזירה מיקרופלואידי לבדיקת גזירת נוזל חד-תאי

To perform the Single-Cell Shear Assay Protocol, use the preassembled shear apparatus setup consisting of a fitted microfluidic flow chamber placed on a Petri dish containing the cells of interest. Place the whole setup onto an inverted microscope with high enough objective and a display monitor. Focus the microscope objective, ensuring adequate contrast and distinct cell edges.Move the microscope stage to ensure the cells are clearly visible on the display monitor and are live images. Select one cell or multiple distinct cells within the imaging or flow path of the fitted flow chamber and the Petri dish. To maintain a continuous uniform viscous media flow, select similar infusion and withdrawal rates.Ensure proper laminar flow of the fluid having Reynold's number or RE less than 100. Click Run on the shear pump to inject and withdraw the shear fluid at a continuous rate. Ensure there are no bubbles during fluid infusion.Begin recording a video by clicking Record on the software before the infused shear fluid makes contact with the cell of interest under the microscope. Continue to record for seven minutes or for the desired duration of the stress exposure or until the cell of interest shears off the bottom of the dish. Click Stop Recording on the microscope software when the run is completed as desired.The software automatically saves the images and videos which can be collected using flash drives for further analysis. Extract the images as TIFF files, preferably at one frame per second for facile analysis.

Processing the Single-Cell Shear Assay Imaging Data

To perform digital image correlation, or DIC, using the DIC software, import the images derived from the sheer assay recording to the DIC software, preferably in the TIF file format. For an optimized correlation, select the sum of differential option which tracks the deformation of a new image with respect to the last image. Select a subset size of 31 by 31 pixels with a step size of 20 pixels.Map out the region of interest for a chosen single cell. For an irregular shape, such as a cell, use a polygon mask to map out the cell's geometry. Select arbitrary points within the mapped cell to track deformation.After mapping, choose specific points on the cell, like the nucleus or cytoplasm, to be analyzed by clicking Add a Strain Gauge and drawing out individual strain gauges at points within the defined cellular boundary. Click on Start to begin the strain processing and obtain strain versus time data. The strain time plot generated as a result of this correlation can be exported as a CSV file for further analysis in MATLAB.Double click or right click on the generated strain time plot and select Export Data. DIC of image frames extracted from sheer assay recordings successfully produced strain time data compatible with the creep stress response. The results of studies using the sheer assay technique showed that cancer cells were generally more mechanically compliant and less viscous than normal cells.The stiffness and viscosities of the cells were observed to decrease with increased cancer progression from a normal cell state to a slightly metastatic state and finally to a highly metastatic state. However, the relaxation time for these cell types showed no significant variations.