Silk Proteins as Biomaterial Additives for DMSO-Reduced Cryopreservation

Background: Cryopreservation is a key enabling technology for cell-based therapies and regenerative medicine; however, the toxicity associated with permeating cryoprotective agents such as dimethyl sulfoxide (DMSO) remains a major limitation, particularly for applications requiring repeated cell administration or long-term storage. Methods: In this study, silk-derived proteins, namely silk fibroin and silk sericin, were investigated as biomaterial-based cryoprotective additives to enable DMSO-sparing cryopreservation strategies. Mouse fibroblasts (3T3) were cryopreserved at -80 °C using conventional DMSO-based media, silk-only formulations, and hybrid formulations combining silk proteins with reduced DMSO concentrations. Post-thaw cell adhesion, metabolic activity, membrane integrity, and cytoskeletal organization were systematically evaluated over a 7-day culture period. Results: Complete replacement of DMSO with silk proteins was insufficient to ensure cell survival, confirming the essential role of permeating cryoprotectants for intracellular protection. In contrast, formulations combining silk fibroin or sericin with 5% (v/v) DMSO supported robust post-thaw viability, preserved cytoskeletal architecture, and promoted favorable recovery kinetics, with cell viability consistently exceeding established biocompatibility thresholds and higher than samples with DMSO alone. Conclusions: These findings support the integration of biomaterial-based components into hybrid cryopreservation formulations and provide design principles relevant to the preservation of more complex multicellular systems.