Cryoprotektants

Cryoprotectants, or cryoprotective agents (CPAs), are substances used to protect biological samples from damage caused by freezing during cryopreservation. They work by reducing ice crystal formation, stabilizing cell membranes and proteins, and mitigating osmotic and mechanical stresses associated with cooling and thawing, enabling long-term storage at low temperatures.

CPAs are broadly categorized as penetrating (permeating) and non-penetrating (non-permeating). Permeating CPAs, such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol, enter cells to protect intracellular components. Non-penetrating CPAs, including disaccharides like sucrose and trehalose, and polymers such as dextran and Ficoll, act extracellularly to limit ice formation and balance osmotic pressures.

Mechanisms include colligative effects that lower the freezing point, promotion of vitrification at high concentrations with rapid cooling to form a glass-like matrix that avoids ice crystals, and stabilization of membranes and proteins during dehydration and rehydration. Some CPAs also reduce oxidative stress and may influence intracellular water dynamics.

Applications include cryopreservation of cells, tissues, embryos, sperm, and, experimentally, organs. Toxicity and osmotic injury are key limitations; cytotoxicity increases with concentration and exposure time and varies by cell type. Protocols balance CPA type and concentration with cooling and warming rates, and removal of CPAs after thawing is often required to recover viability.