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.

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.

Cytocompatibility Evaluation of PEG-Methylsulfone Hydrogels.

Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide-thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide-thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide-thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.