cryopreparation

Cryopreparation refers to the set of procedures applied to biological samples to enable their preservation at cryogenic temperatures. It encompasses sample selection and handling, preconditioning, cleaning or sterilization of surfaces, and the choice and administration of cryoprotective agents. The goal is to minimize injury from dehydration, ice formation, osmotic stress, and toxic effects of cryoprotectants while maintaining biological function after thawing.

Cryoprotectants are substances that reduce ice crystal formation and osmotic stress. Permeating agents such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol penetrate cells to lower intracellular ice formation, while non-permeating agents like sugars and macromolecules control extracellular ice and osmotic balance. Careful optimization of concentration and exposure time is required to balance toxicity against protection.

Equilibration and cooling are key steps. Samples are placed in appropriate containers and subjected to controlled-rate freezing to reach target temperatures gradually, which minimizes intracellular ice. Some protocols use vitrification, a rapid cooling method that forms a glass-like solid without crystalline ice, demanding high cryoprotectant concentrations.

Storage is typically in liquid nitrogen at -196°C, either in liquid or vapor phase, enabling long-term maintenance. Thawing must be rapid to limit recrystallization; post-thaw steps include removal of cryoprotectants, washing, and quality assessment of viability and function.

Applications include storage of mammalian cells, stem cells, embryos, gametes, tissue samples, and cell banks for research, clinical therapies, and agriculture. The field emphasizes standardized quality control, traceability, and regulatory compliance.

Risks and challenges include cryoprotectant toxicity, osmotic injury, and ice recrystallization. Contamination remains a concern. Ongoing research aims to optimize protocols for different materials and improve post-thaw function.