High throughput cell mechanotyping of cell response to cytoskeletal modulations using a microfluidic cell deformation system

Abstract

Cellular mechanical properties influence cellular functions across pathological and physiological systems. The observation of these mechanical properties is limited in part by methods with a low throughput of acquisition or with low accessibility. To overcome these limitations, we have designed, developed, validated, and optimized a microfluidic cellular deformation system (MCDS) capable of mechanotyping suspended cells on a population level at a high throughput rate of ~300 cells per second. The MCDS provides researchers with a viable method for efficiently quantifying cellular mechanical properties towards defining prognostic implications of mechanical changes in pathology or screening drugs to modulate cytoskeletal integrity.

Keywords: cytoskeleton; mechanotyping; microfluidic.

Figure 1:. Graphical overview of MCDS fabrication, setup, and mechanism of action.

A) Representation of the methods used for the fabrication of the MCDS positive mold, the PDMS casting of the mold, and the irreversible bonding of the device to a glass coverslip. B) Graphical representation of the system setup. C) An up-close look at the box highlighted in B, detailing the device components and tubing setup. D) A graphical representation of the cell morphology at different distances within the deformation region of the device. E) A SolidWorks FlowView representation of flow velocity and streamlines within the MCDS.

Figure 2:. Cellular deformation of MDA-MB-231 cells is detectable in MCDS:

A) MDA-MB-231 cells suspended in deformation fluid images on the MCDS microscope. B) A single representative image of an MDA-MB-231 cell being flowed through the channel, where positions of the cell within the channel are stitched together to show deformation at each region within the channel. C-E) Comparison of suspended and deformed cells using quantifiable metrics previously used in deformability cytometry (n = 125). F) Calculated volume for cells in suspension and cells in the deforming region of the MCDS; assuming spheres and ellipsoidal bodies (n = 125). G) Dot plot showing the resulting deformation given the area of MDA-MB-231 cells within the MCDS (n = 500). H) Velocity of MDA-MB-231 cells traveling through the device, calculated by hand, compared to the theoretical velocity calculated from the pump’s flow rates.

Figure 3:. Optimizing MCDS parameters to achieve viable sample collection for efficient analysis.

A) The deformation of MDA-MB-231 cells given the flow rates in μL/hour of the cellular and deformation fluid inlets; these are represented as the first & second numbers for each group on the x-axis (n= 125). B) Location of the center of mass of analyzed MDA-MB-231 cells within the deforming region given flow rates in μL/hour (n = 125). C) Deformation of MDA-MB-231 cells in deformation fluids of varied viscosities created by adding methylcellulose at different weight percentages. The 1% methylcellulose column is labeled with no data, as cells entirely ruptured upon entering the channel, thus no data could be collected (n = 125). D) Deformation of MDA-MB-231 cells given time after the initiation of cell and deformation flow (n = 25). E) Representative images of cells in focus within the deforming region of the MCDS to ensure consistent data acquisition for reproducible analysis. The green box represents the foci chosen for subsequent experiments; however, the black crisp halo would also be a reasonable choice for consistent analysis.

Figure 4:. Validation of MCDS with predefined treatments for stiffness:

A) Deformability of MDA-MB-231 cells given treatment with Vehicle, Taxol (1 μM), Y27632 (20 μM), Blebbistatin (50 μM), and Calyculin-A (100 nM) (n=200). C) Deformability of non-tumorigenic MCF10A, non-metastatic MCF7, and highly metastatic MDA-MB-231 cells. (n=125). B & D) Representative images from each treatment or cell line with a deformability at approximately the average deformability for that cell type. Scale bar = 10 μm. ****p<0.0001

Figure 5:. Detyrosinated microtubules reduce cell deformability.

A) Western blot of deTyr-, Tyr-, and β-tubulin for control and VASH1-overexpressing cells treated with SVC-02. B) Graphical representations of the fold change in deTyr tubulin fluorescence intensities from western blotting normalized to its respective β-tubulin intensity (t-test, **: p<0.01). C) Deformation of MDA-MB-231 control and VASH1-overexpressing cells treated with SVC-02 (t-test, ****: p<0.0001).