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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Cancer Research

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

Published: May 19th, 2023

DOI:

10.3791/65333

1Department of Aerospace and Mechanical Engineering, University of Notre Dame, 2Departments of Mechanical and Biomedical Engineering, Worcester Polytechnic Institute
* These authors contributed equally

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 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.

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 me....

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1. Preparation for the single-cell shear assay

  1. Cell culture
    1. Seed approximately 50,000 suspended single cells in a 35 mm x 10 mm Petri dish containing 2 mL of culture media.
      NOTE: Vortex the suspended cells prior to seeding to break apart cell aggregates.
    2. Incubate the cells at 37 °C and allow between 10 to 48 h for cell attachment and complete cytoskeletal protein formation.
      ​NOTE: Consider the duration of cellular attachment, as well as prolif.......

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The shear assay protocol coupled with deformation analysis using DIC and a viscoelastic model is successful in quantifying the mechanical properties of a single cell in vitro. This method has been tested on human and murine cell lines, including normal human breast cells (MCF-10A)3,4,9, less metastatic triple-negative breast cancer cells (MDA-MB-468)3, triple-negative breast cancer cells (MDA-MB-.......

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The shear assay method, which includes setting up an pseudo-mechanobiological environment to simulate the interaction of cells with the surrounding mechanical microenvironment and their responses to mechanical stresses, has produced a catalog of cellular mechanical properties, whose patterns show conserved physical atypia among cancerous cell lines3,4,5,7,8. T.......

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The authors thank previous researchers from the Soboyejo group at Worcester Polytechnic Institute who first pioneered this technique: Drs. Yifang Cao, Jingjie Hu, and Vanessa Uzonwanne. This work was supported by the National Cancer Institute (NIH/NCI K22 CA258410 to M.D.). Figures were created with BioRender.com.

....

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NameCompanyCatalog NumberComments
CELL CULTURE
.25% Trypsin, 2.21 mM EDTA, 1x[-] sodium bicarbonateCorning25-053-ciFor cellular detachment from substrate in cell culture
15 mL centrifuge tubesFalcon by Corning05-527-90
35 mm Petri dishesCorning430165
50 mL centrifuge tubesFalcon by Corning14-432-22
centrifugeanyFor sterile cell culture
Dulbecco's Modification of Eagle's Medium (DMEM) 1xCorning10-013-cvOr any other media for culturing cells. DMEM was used for culturing U87 cells
glovesanyFor sterile cell culture
Heracell Vios 160i CO2 IncubatorThermo Scientific51033770For Incubation during cell culture
HoodanyFor sterile cell culture
micropipetteanyFor sterile cell culture
micropipette tipsanyFor sterile cell culture
MicroscopeLeica/anyFor sterile cell culture
Phosphate Buffered Saline without calcium and magnesium PBS, 1xCorning21-040-CM
pipetmananyFor sterile cell culture
pipette tipsanyFor sterile cell culture
Precision GP 10 liquid incubatorThermo ScientificTSGP02
T25 flaskCorning430639
T75 flaskCorning430641U
SHEAR ASSAY
100 mL beakeranyFor creating DMEM + methyl cellulose viscous shear media
DMEMCorning
Flow chamber + rubber gasketGlycotech31-001Circular Flow chamber Kit ( for 35 mm tissue culture dishes)
Hybrid RheometerHR-2 Discovery Hybrid RheometerFor determination of shear fluid viscosity
magnetic stir baranyFor creating DMEM + methyl cellulose viscous shear media
magnetic stir plateanyFor creating DMEM + methyl cellulose viscous shear media
methyl celluloseanyTo increase viscosity of DMEM in flow media
Syringe PumpKD Scientific Geminin 88 plus788088For programming fluid infusion and withdrawal
syringes, tubing, and connectorsFor shear apparatus setup
SOFTWARE
ABAQUS softwareSimulia
Digitial Image Correlation softwareLaVision, GermanyDAVIS 10.1.2
Imaging softwareLeica/any microscope software
MATLABMATLABMATLAB_R2020B

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