Trehalose is a naturally occurring, non-reducing disaccharide that has emerged as a leading alternative to conventional toxic cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol. Its unique biochemical properties and ability to protect biological structures during freezing and desiccation have made it a focus of modern cryopreservation research.

Mechanism of Cryoprotection

Trehalose functions primarily as a non-penetrating cryoprotectant (CPA), meaning it remains in the extracellular medium and is not efficiently transported across the cell membrane. Its protective effects are attributed to several complementary mechanisms:

• Water Replacement Hypothesis: Trehalose forms multiple hydrogen bonds with the polar head groups of membrane phospholipids and with proteins. By replacing water molecules that are normally solvating these structures, trehalose stabilizes the lipid bilayer and maintains the native conformation of proteins during dehydration and freezing.
• Vitrification Hypothesis: Trehalose is an excellent glass-forming agent. As water is removed or frozen, trehalose forms an amorphous, glassy matrix that immobilizes biological molecules and prevents the mechanical damage caused by ice crystal growth.
• Ice Growth Inhibition: Unlike some CPAs that primarily work by penetrating cells, trehalose acts as a potent inhibitor of ice crystal growth during freezing and, critically, of ice recrystallization during thawing—a process that causes significant mechanical damage.
• Membrane Stabilization: By forming strong hydrogen bonds with water and membrane components, trehalose helps prevent phase separation and fusion of membranes during freezing stress.