Multi-oscillation microrheology via acoustic force spectroscopy enables frequency-dependent measurements on endothelial cells at high-throughput.

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.