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 signaling axes such as YAP-TAZ. However, the molecular toolbox to achieve mechanotransduction remains virtually limited to non-covalent bonds, which limits the ability to program engineered living matter. Here, it is demonstrated that on-cell chemistry can be leveraged to covalently tether biomaterials directly onto cells, that reveal mechanotransduction via intracellular biophysical programming. Specifically, droplet microfluidics is used to produce single-cell microgels in which individual stem cells are covalently tethered to either soft or stiff hydrogels via on-cell oxidative phenolic coupling. Investigation of mechanotransduction effects at single-cell resolution reveals altered intracellular molecular crowding, calcium signaling, and chromatin organization by regulating cytoplasmic and nuclear volume in a stiffness-dependent yet YAP/TAZ-independent manner. Notably, the addition of conventional dynamic adhesive ligands such as RGDs decreases the chondrogenic commitment of stem cells indicating that covalent cell-material tethering is both efficient and sufficient for programming cell fate. Hence, encoding biomaterials onto cells using covalent on-cell chemistry to attain mechanotransduction expands the ability to guide cellular behavior, which can accelerate the development of in vitro drug-screening models, biofabrication of lab-grown meat, and engineered tissues.