Work

Rational engineering of an antifreeze-based biomaterial with dual-enhanced ice-binding and delivery for high-efficiency cryopreservation

Year: 2025

Type: article

Abstract: INTRODUCTION: Reliable cell cryopreservation is essential for biomedical research and cell-based therapies. Although dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant, its cytotoxicity and clinical incompatibility limit its application, creating an urgent need for safer alternatives. Antifreeze proteins (AFPs) offer favorable biocompatibility and reduced cytotoxicity, making them promising candidates for next-generation cryoprotectants. OBJECTIVES: This study aims to develop a high-performance cryoprotective biomaterial via rational engineering of snow flea AFP (SfAFP), establish an effective DMSO-free cryopreservation strategy, and verify its applicability across multiple cell lines. METHODS: linker (yielding rTAT-GS-MU), whose localization was confirmed by confocal microscopy. Cryopreservation efficacy was tested in EA.hy926, A549, and HEK293 cells. RESULTS: The rSfAFP-MU mutant demonstrated significantly enhanced thermal hysteresis (5 °C vs. 2.19 °C for wild-type SfAFP) and increased EA.hy926 post-thaw recovery by 10.20 % compared to SfAFP. Molecular dynamics simulations revealed that enhanced ice-binding affinity stems from optimized hydrophobic surface intersection angles between monomers, a finding validated by activity reductions in targeted mutants. Confocal imaging confirmed efficient intracellular uptake of rTAT-GS-MU, which correlated with improved cryopreservation efficacy. rTAT-GS-MU achieved 74.77 % EA.hy926 post-thaw recovery (comparable to DMSO) with improved mitochondrial and nuclear DNA integrity and demonstrated efficacy in A549 and HEK293 cells. CONCLUSION: Through rational design, we converted a natural AFP into a high-performance bioactive material that enables efficient cryopreservation of multiple cell lines. This DMSO-free strategy addresses key limitations of traditional methods, offering a valuable alternative for biomedical use.

Source: Journal of Advanced Research

DOI: 10.1016/j.jare.2025.12.008

Authors: Yishan Fu, Yangyang Li, Fei Pan, Sahibzada Muhammad Aqeel, Song Liu, Jian Chen

Institutions: Jiangnan University, Chinese Academy of Agricultural Sciences, Institute of Apiculture Research

Language: English

Topic: nanoparticles nucleation surface interactions

Subfield: Atmospheric Science

Field: Earth and Planetary Sciences

Domain: Physical Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $4,000