High-Throughput Single Cell Mechanomics

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Summary. Phenotypic heterogeneity in cellular bulk populations can result in consequential differences in their response to physical as well as biochemical stimuli. To assess heterogeneity at single cell resolution, several methods have been developed, yet true predictability of cells’ future behavior cannot be reliably determined. To address this challenge, the proposed work will develop a new technological approach to solve the bulk cell heterogeneity problem coined as ‘single cell mechanomics’. This technology will record compression induced dynamic signaling response of single cells to predict and/or drive their future behavior. The technological innovation consists of a ‘smart’ microfluidic device with light actuated microtraps that can capture and compress single cells, and concurrently assess their signaling response, before releasing and capturing each individual cells for subsequent downstream monoclonal culture and analysis. To prove feasibility of this technology, human mesenchymal stromal cells (MSCs) will be used as a representative mechanoresponsive and highly heterogeneous cell type. Aim 1 will design and develop ‘smart’ microfluidic devices with light-actuated mictraps, while Aim 2 will establish a framework to predict and/or drive single cells’ phenotypic outcome based on calcium oscillation dynamics of mechanically compressed single cells. Multivariate predictive analyses will be used to identify relationships between compressive stimuli, calcium signaling, and phenotypic outcome. New relationships derived from this work will be used to identify and sort target cell populations based on their future phenotypes. At present, there is no demonstration of such a technology in the literature. This aligns with the high-risk requirements of this R21 solicitation of having significant future impact.