Cryoprotectant

Cryoprotectants are substances used to protect biological tissue during freezing and thawing. They function mainly by reducing ice formation and growth, lowering the likelihood of lethal intracellular ice, and, in vitrification protocols, promoting a glassy state that suspends molecular motion without ice crystallization. They also help mitigate osmotic stress during addition and removal.

Cryoprotectants are typically categorized as permeating (penetrating) or non-permeating. Permeating CPAs, such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, and 1,2-propanediol, enter cells and replace water to some extent during freezing. Non-permeating CPAs, including sugars such as sucrose and trehalose, as well as mannitol, act outside cells to draw water out and to increase extracellular solute concentration, reducing ice formation and protecting membranes.

In practice, CPAs are used in cryopreservation of a wide range of biological material, including sperm, oocytes, embryos, blood cells, stem cells, and tissue grafts. They are also employed in plant cryopreservation and in biobanking. Vitrification-based approaches rely on high CPA concentrations combined with ultra-rapid cooling to avoid ice crystallization altogether, whereas slow-freezing methods rely on controlled cooling with more moderate CPA concentrations.

Toxicity and osmotic effects are important considerations. Many CPAs are inherently cytotoxic at high concentrations or with prolonged exposure, so protocols optimize the choice and combination of CPAs, their concentrations, and the steps used to add and remove them to minimize damage.