Covalent on-cell conjugation of biomaterials through oxidative phenolic coupling regulates stem cell fate via intracellular biophysical programming

Mechanotransduction is widely used to guide cell fate in hydrogels. Traditionally, hydrogels contain adhesive ligands that dynamically bond with cells to stimulate biochemical signalling axis such as YAP-TAZ. However, the molecular toolbox to achieve mechanotransduction has remained virtually limited to non-covalent bonds, which limits our ability to program engineered living matter. Here, we demonstrate that on-cell chemistry can be leveraged to covalently tether biomaterials directly onto cells, and reveal that mechanotransduction is enabled via intracellular biophysical programming. Specifically, droplet microfluidics produced single-cell microgels in which individual stem cells were extracellularly conjugated to either soft or stiff hydrogels via on-cell oxidative phenolic coupling, which allowed for investigation of mechanotransduction at single-cell resolution. Interestingly, this altered intracellular molecular crowding, calcium signalling, and chromatin organization by regulating cytoplasmic and nuclear volume in a stiffness-dependent yet YAP/TAZ-independent manner. Notably, addition of conventional dynamic adhesive ligands such as RGDs decreased chondrogenic commitment indicating that covalent cell-material tethering is both efficient and sufficient for programming cell fate. Encoding biomaterials with a novel form of mechanotransduction in the form of covalent on-cell chemistry, such as oxidative phenolic coupling, expands our ability to guide cellular behaviour, which can accelerate development of drug-screening models, lab-grown meat, and engineered tissues.