Monolayer Stress Microscopy: Developing 3D, Elastographic, and High-Throughput Capabilities

The collective transmission of forces in multicellular colonies plays an important role in biological processes such as development, endothelial function and wound healing. The quantification of intracellular forces by monolayer stress microscopy (MSM) has improved our understanding of these phenomena, and provides great promise for the functional screening of contractile tissues in the context of drug development. However, current MSM methods present a number of limitations: they assume that the monolayer only deforms laterally, introduce boundary artifacts, and are restricted to low-throughput applications. This dissertation addresses these limitations and provides the first high-throughput quantification of three-dimensional (3D) intracellular stresses in applications relevant to biology and biotechnology, ranging from leukocyte extravasation to high-throughput functional screening of cardiomyocytes. We introduce a 3D-MSM method that incorporates for the first time monolayer bending. 3D-MSM measures the 3D traction stresses exerted by the cells on a flexible substrate and calculates intracellular stresses by imposing equilibrium of forces and moments in the monolayer. We present 3D-MSM measurements in micropatterned cell islands of different sizes and shapes. We show that intracellular stresses caused by lateral deformation are transmitted across long distances, whereas bending-induced stresses remain confined to 1-2 cell lengths from bending sites. We hypothesize that the localized effect of bending-induced stresses may be important in processes such as cellular extravasation, which are accompanied by large 3D deflections of the endothelium and require localized permeability changes. Overall, these results suggest that bending and lateral stresses can propagate mechanical signals at different length scales, and reveal that the transmission of forces across cell junctions is more three-dimensional and complex than previously believed. We also present a novel high-throughput functional screening assay that quantifies cardiomyocyte force generation and stiffness changes during the cardiac cycle. The method jointly measures the intracellular stresses within cardiomyocyte monolayers and their elastic modulus. The main novelty of this method is that it utilizes the forces generated by the cells themselves to probe their material properties and is, thus, fully non-invasive. We present the proof-of-concept application of our method with dose-response measurements of a series of benchmark compounds tested on monolayers of hiPSC cardiomyocytes