Little is known about how mitotic cells round against epithelial confinement. At the beginning of mitosis, cells markedly change their morphology as they round up 1. During mitotic cell rounding, the microtubule cytoskeleton forms the mitotic spindle, a central machinery that captures and organizes chromosomes 2 . Mitotic cell rounding occurs in the vast majority of animal cells 3 and plays a role in maintaining tissue organization. Here, we have engineered micropillar arrays with spatial and mechanical properties to mimic the mechanical confinement of epithelia and to measure the forces generated by single epithelial cells rounding for mitosis. For our studies we use Madin?Darby canine kidney (MDCK) cell lines that are commonly used as a model for epithelial cells. We observe that MDCK cells deflect nearby micropillars as they round and thereby create sufficient space for mitosis. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells 4,5 . The availability of large pillar arrays and the optical readout of the assay enables the simultaneous characterization of hundreds of single cells to investigate the cellular mechanisms that drive the drastic shape change in mitosis. We have demonstrated that the contributions of proteins involved in mitotic cell rounding to generating force can be quantified by combining the micropillar assay with targeted chemical perturbation. Thus, the micropillar assay opens up the possibility to perform a large-scale screen for genes and mechanisms involved in mitotic cell rounding. References: 1. McConnell, C. H. Mitosis in hydra - mitosis in the ectodermal epitheliomuscular cells of Hydra. Biol. Bull. 64, 86?95 (1933). 2. Cramer, L. P. & Mitchison, T. J. Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding. Mol. Biol. Cell 8, 109?119 (1997). 3. Kline-Smith, S. L. & Walczak, C. E. Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics. Mol. Cell 15, 317?327 (2004). 4. Stewart, M. P. et al. Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469, 226?230 (2011). 5. Sorce, B. et al. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement. Nature Commun. 25, 8872-8876 (2015)