Poster P7 - Indentation Measurement of Cell Mechanical Anisotropy Using a Toroidal Probe

Introduction: The mechanical properties of living cells reflect their physiological state and may serve as a hallmark for diseases such as cancer, calcification, and atherosclerosis. While many cell types are highly polarized in their structure, the mechanical anisotropy of the cells is often neglected in mechanical analysis. In most studies, an isotropic elastic modulus is used to report or describe the mechanical properties of the cell. This oversimplification may bias the interpretation in cell mechanics and mechanobiology.
Understanding mechanical anisotropy of cells is hindered by a lack of accessible methods for measurement. Indentation is the most common method for measuring stiffness of cells, yet conventional indenters are equipped with spherical, conical, or pyramidal probes which do not yield information related to mechanical anisotropy. Alternative approaches such as magnetic twisting cytometry, magnetic tweezers, and micro biaxial stretch generally require custom-made devices or complex image analysis of the 3D deformation fields which are not accessible to most researchers. A straight-forward indentation method for the measurement of anisotropic stiffness of cells is needed.
Here, we propose a method to measure anisotropic stiffness of cells by two nano-indentations in orthogonal directions using a probe mounted with a novel toroidal tip. We nano-3D printed probe tips with 10:1 aspect ratio and attached the tip to a commercial indenter probe, then indented aligned and isotropic cell monolayer and single polarized cells using a commercial nano-indenter equipped with this probe to demonstrate the applicability of this method.

Materials and

Methods: The toroidal probe tip was designed to be 10 µm in major radius and 1 µm in minor radius. The tip was 3D printed by two-photon polymerization in 50 nm resolution using a NanoScribe GT+ 3D printer. Then the tip was mounted to a tipless nanoindenter probe (Optics11). (Fig. 1).
Anisotropic porcine aortic valvular interstitial cell (PAVIC) monolayers and isotropic PC-9 cell monolayers were cultured in 35-mm cell culture petri dishes with parallel scratches at the bottom (Fig. 2A). Single PAVICs were also cultured in 35-mm dishes.
Indentation tests were performed using an Optics11 Chiaro nano-indenter equipped with the toroidal tip probe mounted onto an optical microscope. The cell monolayers were randomly indented at 15-25 spots in the direction parallel to and across the scratch. For single cells, the indentation was performed on both nucleus and cytosol along the directions parallel and perpendicular to the cell long axis. The indentation speed was set to 500 nm/s, with 500 nm maximum indentation depth. The order of indentation was randomized. The force-indentation curves were fit to the Hertzian model for elliptical contact, in which the effective Young’s modulus (E*) is calculated based on the force, indentation depth, and probe geometry. Effective Young’s moduli along and across the cell long axis were compared with a t-test with p< 0.05 considered significant.

Results, Conclusions, and Discussions: Results
Mechanical anisotropy was found on all eight PAVIC monolayers, with an average effective Young’s modulus of 10.48±1.44 kPa in indentation parallel to the scratch and 24.47±3.87 kPa in indentation perpendicular to the scratch (p < 0.001). Anisotropic indentation on all three PC-9 monolayers did not find mechanical anisotropy, with an average effective Young’s modulus of 2.12±0.17 kPa in the parallel direction and 2.25 ±0.25 kPa in the perpendicular direction (p=0.63) (Fig. 3A).
Anisotropic indentation on both the nucleus and cytosol of the individual PAVICs yielded a higher effective Young’s modulus when indenting along the perpendicular direction (p < 0.001) (Fig. 3B). Indentation on the cell nucleus yielded an effective Young’s modulus of 10.75±3.35 kPa in the parallel direction and 17.25±3.35 kPa in the perpendicular direction. The indentation on the cytosol yielded an effective Young’s modulus of 12.02±4.51 kPa in the parallel direction and 17.91±5.87 kPa in the perpendicular direction.

Discussion & Conclusion
Despite the importance of cell polarity and cytoskeletal alignment, cell mechanical anisotropy is rarely measured experimentally due to technical difficulties. Here, we present a facile method to measure the mechanical anisotropy of cells by making two orthogonal indentations with a toroidal probe without the need for image analysis. Our methods can be implemented with most commercially available indenters and AFMs for a cost of ~$20 and the tip can be printed and mounted in less than four hours.
In this study, we measured the mechanical anisotropy in aligned and non-aligned cell monolayers. As expected, we found the aligned cell monolayer to be mechanically anisotropic, while the non-aligned cell monolayer to be mechanically isotropic. The indentation on both cytosol and nucleus of PAVICs shows mechanical anisotropy with similar modulus. The mechanical anisotropy of the cells may be attributed to the aligned F-actin.
One limitation of this study is that the modulus is calculated based on Hertzian contact theory which assumes material isotropy. In the next step of the study, we plan to apply an analytical model to directly extract the anisotropic mechanical parameters from the indentation curves. We expect this method will facilitate our understanding of cell mechanics and mechanobiology.

Acknowledgements (Optional): This work was supported by the NSF (CMMI 1761432) and ARMI BiofabUSA (T0137).