Nanonet Force Microscopy for Measuring Cell Forces.

The influence of physical forces exerted by or felt by cells on cell shape, migration, and cytoskeleton arrangement is now widely acknowledged and hypothesized to occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Our previous work using the non-electrospinning Spinneret-based Tunable Engineered Parameters' suspended fibers has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to focal adhesion cluster lengths. Here, we present the development and application of a suspended nanonet platform for measuring C2C12 mouse myoblast forces attached to fibers of three diameters (250, 400, and 800 nm) representing a wide range of structural stiffness (3-50 nN/μm). The nanonet force microscopy platform measures cell adhesion forces in response to symmetric and asymmetric external perturbation in single and cyclic modes. We find that contractility-based, inside-out forces are evenly distributed at the edges of the cell, and that forces are dependent on fiber structural stiffness. Additionally, external perturbation in symmetric and asymmetric modes biases cell-fiber failure location without affecting the outside-in forces of cell-fiber adhesion. We then extend the platform to measure forces of (1) cell-cell junctions, (2) single cells undergoing cyclic perturbation in the presence of drugs, and (3) cancerous single-cells transitioning from a blebbing to a pseudopodial morphology.