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

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 cancerous and non-cancerous 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 field of research is the use of microfluidic devices, and high-throughput techniques.

So, on the study, 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, scale and 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 stresses, and the extent to which resist mechanical stress is dependent on structural integrity, which is dictated to 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 assay technique we'll 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.

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 stirrer or hot plate. While continuously stirring the media, gently add methyl cellulose into it to help the methyl cellular 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 desire to 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 preparative 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 one 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. 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 a 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.

To perform digital image correlation or DIC using the DIC software, import the images derived from the shear assay recording to the DIC software, preferably in the TIFF 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 x 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 shear assay recordings successfully produced strain time data compatible with the creep stress response. The results of studies using the shear 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.