Abstract
Active microrheology is one of the main methods to determine the mechanical properties of cells and tissue, and the modelling of these viscoelastic properties is under heavy debate with many competing approaches. Most experimental methods of active microrheology such as optical tweezers or atomic force microscopy based approaches rely on single cell measurements, and thus suffer from a low throughput. Here, we present a novel method for frequency-dependent microrheology on cells using acoustic forces which allows multiplexed measurements of several cells in parallel. Acoustic force spectroscopy (AFS) is used to generate multi-oscillatory forces in the range of pN–nN on particles attached to primary human umbilical vein endothelial cells (HUVEC) cultivated inside a microfluidic chip. While the AFS was introduced as a single-molecule technique to measure mechanochemical properties of biomolecules, we exploit the AFS to measure the dynamic viscoelastic properties of cells exposed to different conditions, such as flow shear stresses or drug injections. By controlling the force and measuring the position of the particle, the complex shear modulus G*(ω) can be measured continuously over several hours. The resulting power-law shear moduli are consistent with fractional viscoelastic models. In our experiments we confirm a decrease in shear modulus after perturbing the actin cytoskeleton via cytochalasin B. This effect was reversible after washing out the drug. Additionally, we include critical information for the usage of the new method AFS as a measurement tool showing its capabilities and limitations and we find that for performing viscoelastic measurements with the AFS, a thorough calibration and careful data analysis is crucial, for which we provide protocols and guidelines.
Highlights
The mechanical properties of cells and tissues are closely related to their biological function, and defects in stiffness and viscosity have been related to several malfunctions and diseases. 1–4 An example of cells highly exposed to variable mechanical forces are endothelial cells (ECs) which make up the inner wall of blood vessels
We establish a novel method for multi-oscillation microrheology on cell monolayers using acoustic forces with the Acoustic Force Spectroscopy (AFS) which alled us to determine the frequency dependent viscoelastic properties of human umbilical vein endothelial cells (HUVEC) monolayers
While the advantage of high-throughput measurements does not apply for the present microrheological experiment, the advantage of the flow system enabled the first measurement of the dynamics of the complex shear modulus of HUVEC monolayers under flow, and to determine the time evolution of a drug effect on these cells
Summary
The mechanical properties of cells and tissues are closely related to their biological function, and defects in stiffness and viscosity have been related to several malfunctions and diseases. 1–4 An example of cells highly exposed to variable mechanical forces are endothelial cells (ECs) which make up the inner wall of blood vessels. 1–4 An example of cells highly exposed to variable mechanical forces are endothelial cells (ECs) which make up the inner wall of blood vessels. They are constantly exposed to variable shear stresses originating from the blood flow. These forces are known to be important regulators for proper EC function. 3,9 The cell mechanical response to different exposures, such as biochemical stimuli, is an important characterization to better understand the processes in healthy and malfunctional cells. 13 Recently, a fractional viscoelastic element has turned out to be a highly flexible approach to describe the different experimental data measured in microrheology.
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