Hyaluronic acid and zinc ion-based cryoprotective complex for enhancing post-thaw cell viability

Cryopreservation is essential for the long-term storage and transport of cells, tissues, and biological products. However, freezing and thawing can damage cells through ice crystal formation, osmotic stress, and dehydration, reducing post-thaw viability and cellular function. Conventional cryoprotective agents such as dimethyl sulfoxide and glycerol are commonly used to minimize cryoinjury, but their cytotoxicity, osmotic imbalance, and adverse effects on cell physiology remain major limitations. To overcome these challenges, we developed a hyaluronic acid- and zinc ion (Zn²⁺)-based cryoprotective material designed to protect cells from extracellular ice formation and membrane damage during freezing and thawing. Hyaluronic acid was employed for its high water-binding capacity and viscosity, which promote strong interactions with water molecules and suppress ice crystal formation during freezing. Zn²⁺ was incorporated to stabilize cellular membranes by maintaining lipid fluidity at low temperatures. To achieve synergistic cryoprotective effects, thiol groups were introduced into the hyaluronic acid backbone to synthesize thiolated hyaluronic acid (HA-SH), which was subsequently complexed with Zn²⁺ via coordination to form HA-SH/Zn²⁺ complexes. These complexes improved the post-thaw viability of human dermal fibroblasts compared with conventional freezing conditions, indicating their potential as cryoprotective additives. Furthermore, HA-SH/Zn²⁺ complexes added to culture media upregulated transforming growth factor-beta 1 and vascular endothelial growth factor, suggesting their efficacy as bioactive additives that promote regenerative cellular responses. This dual functionality supports both long-term cell storage and subsequent cell culture applications of our bioactive complex. Overall, HA-SH/Zn²⁺ complexes represent a promising multifunctional additive for enhancing the efficacy of cryopreservation and cellular bioactivity.