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Abstract Various signals in tissue microenvironments are often unevenly distributed around cells. Cellular responses to asymmetric cell‐matrix adhesion in a 3D space remain generally unclear and are to be studied at the single‐cell resolution. Here, the authors developed a droplet‐based microfluidic approach to manufacture a pure population of single cells in a microscale layer of compartmentalized 3D hydrogel matrices with a tunable spatial presentation of ligands at the subcellular level. Cells elongate with an asymmetric presentation of the integrin adhesion ligand Arg‐Gly‐Asp (RGD), while cells expand isotropically with a symmetric presentation of RGD. Membrane tension is higher on the side of single cells interacting with RGD than on the side without RGD. Finite element analysis shows that a non‐uniform isotropic cell volume expansion model is sufficient to recapitulate the experimental results. At a longer timescale, asymmetric ligand presentation commits mesenchymal stem cells to the osteogenic lineage. Cdc42 is an essential mediator of cell polarization and lineage specification in response to asymmetric cell‐matrix adhesion. This study highlights the utility of precisely controlling 3D ligand presentation around single cells to direct cell polarity for regenerative engineering and medicine. 

Abstract Advances in engineered hydrogels reveal how cells sense and respond to 3D biophysical cues. However, most studies rely on interfacing a population of cells in a tissue‐scale bulk hydrogel, an approach that overlooks the heterogeneity of local matrix deposition around individual cells. A droplet microfluidic technique to deposit a defined amount of 3D hydrogel matrices around single cells independently of material composition, elasticity, and stress relaxation times is developed. Mesenchymal stem cells (MSCs) undergo isotropic volume expansion more rapidly in thinner gels that present an Arg‐Gly‐Asp integrin ligand. Mathematical modeling and experiments show that MSCs experience higher membrane tension as they expand in thinner gels. Furthermore, thinner gels facilitate osteogenic differentiation of MSCs. By modulating ion channels, it is shown that isotropic volume expansion of single cells predicts intracellular tension and stem cell fate. The results suggest the utility of precise microscale gel deposition to control single cell functions.