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

Abstract

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

“… , For comparison and benchmarking, hydrogels with similar compositions were prepared starting from 4-arm-PEG-MAL macromers (Figure ). PEG-MS/SH hydrogels have shown a Young’s moduli of approximately 0.9 kPa after swelling (measured by rheology, 4-arm-PEG-MS and 4-arm-PEG-SH of 20 kDa), a swelling mass of 47 mg of water/mg polymer and a stability of 26 days in DPBS, as previously published. , PEG-MAL/SH hydrogels (4-arm-PEG-MAL, 20 kDa) showed a Young’s modulus of approximately 0.6 kPa (measured by nanoindentation) and a swelling mass of 77 mg of water/mg of polymer …”

“…RGD and VPM peptides were dissolved in HEPES 20 mM supplemented with 8 mg/mL NaHCO 3 . This buffer concentration maintains a constant pH during polymerization in spite of the release of methylsulfonate anions as byproduct of the reaction …”

“…We have recently presented the methylsulfonyl/thiol (MS/SH) reaction as an alternative thiol-based cross-linking system. Thiols react with arylMS groups at physiological conditions (pH range of 6 to 9 and temperature of 37 °C) at high conversion and with good selectivity, without the need of photoactivation. , The thioether reaction product is stable and proven cytocompatible in in vitro experiments. Within physiological conditions, we demonstrated that PEG-SH and PEG-MS macromers at typical concentrations for cell encapsulation form hydrogels within minutes time scale. , In contrast, when the well established MAL/SH reaction is used in these conditions, the hydrogels are formed within seconds time scale at the pH required to achieve high cross-linking. , This can lead to inhomogeneity and low stability in the resulting hydrogels. ,,− We demonstrated that MS/SH cross-linked hydrogels showed higher homogeneity than their MAL/SH counterparts. , …”

“…Thiols react with arylMS groups at physiological conditions (pH range of 6 to 9 and temperature of 37 °C) at high conversion and with good selectivity, without the need of photoactivation. 28 , 29 The thioether reaction product is stable and proven cytocompatible in in vitro experiments. Within physiological conditions, we demonstrated that PEG-SH and PEG-MS macromers at typical concentrations for cell encapsulation form hydrogels within minutes time scale.…”

“…Within physiological conditions, we demonstrated that PEG-SH and PEG-MS macromers at typical concentrations for cell encapsulation form hydrogels within minutes time scale. 28 , 29 In contrast, when the well established MAL/SH reaction is used in these conditions, the hydrogels are formed within seconds time scale at the pH required to achieve high cross-linking. 11 , 30 This can lead to inhomogeneity and low stability in the resulting hydrogels.…”