Thiol-Methylsulfone-Based Hydrogels for Cell Encapsulation: Reactivity Optimization of Aryl-Methylsulfone Substrate for Fine-Tunable Gelation Rate and Improved Stability.

Hydrogels are widely used as hydrated matrices for cell encapsulation in a number of applications, spanning from advanced 3D cultures and tissue models to cell-based therapeutics and tissue engineering. Hydrogel formation in the presence of living cells requires cross-linking reactions that proceed efficiently under close to physiological conditions. Recently, the nucleophilic aromatic substitution of phenyl-oxadiazole (Ox) methylsulfones (MS) by thiols was introduced as a new cross-linking reaction for cell encapsulation. Reported poly(ethylene glycol) (PEG)-based hydrogels featured tunable gelation times within seconds to a few minutes within pH 8.0 to 6.6 and allowed reasonably good mixing with cells. However, their rapid degradation prevented cell cultures to be maintained beyond 1 week. In this Article, we present the reactivity optimization of the heteroaromatic ring of the MS partner to slow down the cross-linking kinetics and the degradability of the derived hydrogels. New MS substrates based on phenyl-tetrazole (Tz) and benzothiazole (Bt) rings, with lower electrophilicity than Ox, were synthesized by simple pathways. When mixed with PEG-thiol, the novel PEG-MS extended the working time of precursor mixtures and allowed longer term cell culture. The Tz-based MS substrate was identified as the best candidate, as it is accessible by simple chemical reactions from cost-effective reactants, hydrogel precursors show good stability in aqueous solution and keep high chemoselectivity for thiols, and the derived Tz gels support cell cultures for >2 weeks. The Tz system also shows tunable gelation kinetics within seconds to hours and allows comfortable manipulation and cell encapsulation. Our findings expand the toolkit of thiol-mediated chemistry for the synthesis of hydrogels with improved properties for laboratory handling and future automatization.

Thiol-Methylsulfone-Based Hydrogels for 3D Cell Encapsulation.

Thiol-maleimide and thiol-vinylsulfone cross-linked hydrogels are widely used systems in 3D culture models, in spite of presenting uncomfortable reaction kinetics for cell encapsulation: too fast (seconds for thiol-maleimide) or too slow (minutes-hours for thiol-vinylsulfone). Here, we introduce the thiol-methylsulfone reaction as alternative cross-linking chemistry for cell encapsulation, particularized for PEG-hydrogels. The thiol-methylsulfone reaction occurs at high conversion and at intermediate reaction speed (seconds-minutes) under physiological pH range. These properties allow easy mixing of hydrogel precursors and cells to render homogeneous cell-laden gels at comfortable experimental time scales. The resulting hydrogels are cytocompatible and show comparable hydrolytic stability to thiol-vinylsulfone gels. They allow direct bioconjugation of thiol-derivatized ligands and tunable degradation kinetics by cross-linking with degradable peptide sequences. 3D cell culture of two cell types, fibroblasts and human umbilical vein endothelial cells (HUVECs), is demonstrated.