Gelation Kinetics and Mechanical Properties of Thiol‐Tetrazole Methylsulfone Hydrogels Designed for Cell Encapsulation

Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol‐tetrazole methylsulfone (TzMS) reaction crosslinks star‐poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG‐based hydrogels formed by thiol‐TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell‐adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG‐4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture. Hydrogels crosslinked with thiol‐tetrazole methylsulfone (TzMS) chemistry form gels within minutes under near‐physiological conditions. Gelation kinetics and mechanical properties are explored in this work as a function of buffer, crosslinker structure and degree of TzMS functionality adjusted by including cell‐adhesive ligand, paving the way for reliable 3D cell culture workflows.