Dynamic Exchange in 3D Cell Culture Hydrogels Based on Crosslinking of Cyclic Thiosulfinates

Dynamic polymer materials are highly valued substrates for 3D cell culture due to their viscoelasticity, a time‐dependent mechanical property that can be tuned to resemble the energy dissipation of native tissues. Herein, we report the coupling of a cyclic thiosulfinate, mono‐S‐oxo‐4‐methyl asparagusic acid, to a 4‐arm PEG‐OH to prepare a disulfide‐based dynamic covalent hydrogel with the addition of 4‐arm PEG‐thiol. Ring opening of the cyclic thiosulfinate by nucleophilic substitution results in the rapid formation of a network showing a viscoelastic fluid‐like behaviour and relaxation rates modulated by thiol content through thiol‐disulfide exchange, whereas its viscoelastic behaviour upon application as a small molecule linear crosslinker is solid‐like. Further introduction of 4‐arm PEG‐vinylsulfone in the network yields a hydrogel with weeks‐long cell culture stability, permitting 3D culture of cell types that lack robust proliferation, such as human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs). These cells display native behaviours such as cell elongation and spontaneous beating as a function of the hydrogel's mechanical properties. We demonstrate that the mode of dynamic cyclic thiosulfinate crosslinker presentation within the network can result in different stress relaxation profiles, opening the door to model tissues with disparate mechanics in 3D cell culture. Cyclic thiosulfinates can generate disulfide‐crosslinked dynamic covalent hydrogels without additional stimuli to gain control over stress relaxation based on thiol content and mode of crosslinker application. Pluripotent stem cell‐derived cardiomyocytes can be 3D cultured in hydrogels containing macromonomers with this chemistry, displaying native behaviours, such as cell spreading and spontaneous beating.