Transient Catalase Immobilization for Cytoprotection during H2O2-Mediated Cell-Laden Hydrogel Fabrication.

Abstract

Hydrogen peroxide (H₂O₂) is widely used in horseradish peroxidase (HRP)-mediated hydrogel cross-linking in the fabrication of cell-laden constructs using phenol group-containing polymers. However, H₂O₂ poses cytotoxic risks at high concentrations. As H₂O₂ also functions as a signaling molecule, its local concentration must be precisely controlled. Here, we report a transient and cytocompatible strategy for immobilizing catalase, an enzyme that decomposes H₂O₂, on cell surfaces via gelatin-mediated electrostatic adsorption to mitigate H₂O₂-induced oxidative stress during subsequent HRP-mediated hydrogel fabrication for cell encapsulation. The surface-bound catalase decomposes excess H₂O₂ near the cell membrane and is gradually released within a few hours. Compared with nontreated cells, catalase-immobilized HeLa and NMuMG cells exhibit 10-20% higher viability and up to 5-fold greater proliferation under exposure to 1-2 mM H₂O₂ for 30 min. Catalase immobilization on the cell surface is compatible with HRP-catalyzed hydrogel cross-linking for cell encapsulation, and while it moderately reduces the bulk stiffness of the resulting hydrogel, the cells encapsulated in the hydrogels retain high viability and proliferative capacity. This method offers a simple, reversible, and biocompatible approach for reducing oxidative cytotoxicity during HRP-mediated hydrogel fabrication for cell encapsulation, supporting its potential utility in biomedical applications, such as tissue engineering and regenerative medicine.