cryoprotection

Cryoprotection refers to methods and agents used to preserve biological material at subzero temperatures by preventing ice formation and the associated damage from ice crystals and osmotic stress. The goal is to minimize mechanical injury from ice, chemical toxicity, and disruption of cellular membranes during freezing and storage. Cryoprotectants are chemical substances that lower the freezing point of water, reduce ice crystallization, and protect cellular structures during cooling and warming. Preservation approaches are broadly categorized as vitrification or slow-freezing, depending on cooling rates and CPA concentrations.

Two broad strategies predominate. Vitrification uses high concentrations of cryoprotectants along with ultra-rapid cooling to transform the solution into a glassy, ice-free state, avoiding crystallization. Slow-freezing employs more moderate CPA levels with controlled cooling rates that allow water to exit cells and form extracellular ice, reducing intracellular ice formation but requiring careful optimization to minimize osmotic and thermal stress.

Common cryoprotectants include permeating agents such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and propylene glycol; and non-permeating agents such as sugars like trehalose and sucrose, as well as polymers. The choice and concentration of CPAs depend on cell type, sample size, and intended use, because many agents exhibit toxicity at higher concentrations.

Applications encompass cryopreservation of cells (including stem cells, blood components, sperm and oocytes), embryos, tissues, and some organs; biobanking of seeds and biological materials; and long-term storage for research and clinical use. Practical challenges include CPA toxicity, osmotic injury, incomplete CPA penetration, ice formation during cooling or warming, and the need for precise storage and warming protocols to maintain viability and function.