Two‐Photon Polymerized 3D Geometries to Study Single‐Cell Mechanotransduction and Force Generation in Mesenchymal Stromal Cells

Understanding cellular response to mechanical cues in three-dimensional (3D) environments remains a central challenge in cell biology. Shape and force distribution are key regulators of mechanosensing. In vivo, cells are embedded in 3D environments where force transmission and cytoskeletal behavior differ markedly from two-dimensional systems. However, current tools lack the resolution to precisely control single-cell geometry or quantify traction forces in defined 3D contexts. Here, a direct laser writing-based platform is presented that fabricates microscale cage structures capable of confining individual mesenchymal stem cells in defined 3D geometries while enabling high-resolution traction force measurements. The system allows independent control of cell volume and shape, and captures nanowire deflection as a readout of cell-generated forces at varying heights. Using this platform, this study reveals that 3D shape alone, modulates cytoskeletal organization, contractility, and localization of the mechanosensitive transcription factor Yes-associated protein (YAP) in a shape and time dependent manner. Square cages induced previously unreported vertical actin fibers and corner-enriched myosin accumulation, suggesting that pointed 3D geometry alters internal force distribution. Delayed YAP nuclear translocation indicates a time-sensitive mechanotransduction response to 3D confinement. Altogether, this platform offers a tunable 3D confinement tool and new insights into how shape alone direct cellular force architecture and mechanosensitive signaling.