High-throughput single cell motility analysis using nanowell-in-microwells.

Cell motility is important to many biological processes including cancer, immune response, and tissue repair. Conventional assays measure bulk cell motility, potentially overlooking important heterogeneity and missing important high motility subpopulations. Here, we introduce a high-throughput single-cell motility assay using nanowell-in-microwell plates to precisely track single cell position and analyze their migratory trajectories. By physically confining individual cells in nanowells, we eliminate cell-cell interactions and simplify cell segmentation and tracking. Using this platform, we characterized the motility of single cells across different culture conditions to identify distinct motility phenotypes. Single-cell trajectory analysis revealed pronounced directional persistence, with cells predominantly maintaining their direction of travel and trajectory along nanowell boundaries. Additionally, our approach facilitates the generation of labeled image datasets suitable for AI models to rapidly identify cell motility phenotypes from single-cell images. Together, our platform provides a robust, scalable method to analyze cell motility phenotypes and migration behavior at single-cell resolution.