Photothermally Powered 3D Microgels Mechanically Regulate Mesenchymal Stem Cells Under Anisotropic Force

Exogenous forces significantly influence mammalian cell behavior, yet current strategies fail to resolve signaling processes between individual cells under conditions that accurately mimic the native microenvironment. This work presents a new cell culture technology capable of applying spatially patterned exogenous forces on individual cells within multicellular clusters encased in three-dimensional (3D) hydrogel matrices. Photothermally powered 3D microgels containing stem cells and integrated force generators are engineered to investigate intercellular communication under anisotropic forces with excellent spatial resolution (≈1 µm). Varying force patterns, such as uniform compression versus spatially heterogeneous tension, are achieved in 3D by relying on the synergistic effect of plasmonic gold nanorods and thermally responsive co-polymers under light actuation. The microgels generate 17-34 nN force locally, which activates mechanically sensitive ion channels in encapsulated cells stimulated with isotropically applied compression and spatially heterogeneous tension in 3D in a selective manner. Spatially patterned exogenous forces trigger F-actin remodeling, nuclear translocation of Yes-associated protein (YAP) and Runt-related transcription factor 2 (RUNX2) in encapsulated cells following cyclic stimulation. Sustained application of exogenous forces over three days is sufficient to regulate stem cell fate toward osteogenesis. This technology allows combinatorial studies of biomolecular and biophysical cues in 3D, making it suitable for applications in mechanobiology and bioengineering.