Cryopreservation is based on the ability of certain small molecules to enter cells and prevent dehydration and formation of intracellular ice crystals, which can cause cell death and destruction of cell organelles during the freezing process. Two common cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol. Glycerol is used primarily for cryoprotection of red blood cells, and DMSO is used for protection of most other cells and tissues. A sugar called trehalose, which occurs in organisms capable of surviving extreme dehydration, is used for freeze-drying methods of cryopreservation. Trehalose stabilizes cell membranes, and it is particularly useful for the preservation of sperm, stem cells, and blood cells.

Most systems of cellular cryopreservation use a controlled-rate freezer. This freezing system delivers liquid nitrogen into a closed chamber into which the cell suspension is placed. Careful monitoring of the rate of freezing helps to prevent rapid cellular dehydration and ice-crystal formation. In general, the cells are taken from room temperature to approximately −90 °C (−130 °F) in a controlled-rate freezer. The frozen cell suspension is then transferred into a liquid-nitrogen freezer maintained at extremely cold temperatures with nitrogen in either the vapour or the liquid phase. Cryopreservation based on freeze-drying does not require use of liquid-nitrogen freezers.