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Best practices for cryopreserving, thawing, recovering, and assessing cells

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Long-term storage of cell stocks insures that cells are available for use whenever needed. Cryopreservation of cells is the method of choice for preservation of important or rare cell stocks. There are several factors to consider when establishing a protocol for freezing, thawing, and recovery of cells after storage. These parameters may include cell concentration, cryoprotectant choice and concentration, and thawing rate among others. Further, the assessment of cell viability and/or function prior to and following cryopreservation is imperative in order to accurately determine downstream utility as well for optimizing the cryopreservation process. This chapter is designed to provide guidance and insight into developing robust and successful protocols for preserving cells that will preserve cell stocks and provide optimal cell yield and viability.

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The author would like to thank the following for their invaluable contributions and review of the manuscript—Gertrude Case Buehring, Eugene Elmore, Raymond W. Nims, Paul Price, Yvonne Reid, and Frank Simione.

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Correspondence to John M. Baust.

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Editor: Tetsuji Okamoto

Glossary of Terms

Air time

the time between removing a sample form storage and placement in to a water bath or other thawing device.


gene-regulated cell death.


of or containing water, typically as a solvent or medium

Cooling rate

the rate at which temperature of a sample decreases with time usually measured in °C/min.


a process where organelles, cells, tissues, organs or any other biological construct are preserved by cooling to very low temperatures (typically − 80°C using solid carbon dioxide or − 196°C using liquid nitrogen) without causing additional damage caused by the formation of ice during freezing.

Cryopreservation induced delayed onset cell death (CIDOCD)

cell death which occurs in a delayed manner 24–72 h post-thaw due to the apoptotic and necrotic activity.

Cryoprotective agent/cryoprotectant

a substance used to protect biological tissue from freezing damage. An example would by dimethyl sulfoxide or glycerol.


the degree to which an agent has a specific destructive action on cells.


an insulated container used especially to store liquefied gases, having a double wall with a vacuum between the walls and silvered surfaces facing the vacuum.

Disaccharide sugar

sugar formed when two monosaccharides (simple sugars) are joined by glycosidic linkage. Like monosaccharides, disaccharides are soluble in water. Three common examples are trehalose, sucrose, and maltose.

Equilibrium freezing point

the temperature at which a given solution will freeze. This can change based on the composition of the solution that is being cooled.

Gel phase

the state of a lipid bilayer or membrane in which the lipids are packed more orderly and remain relatively immobile. This usually occurs below the transition temperature, Tm.


a condition where the core temperature of an organism falls below a critical threshold. In humans, hypothermia occurs when the body temperature drops below 35°C.


a decrease in the availability of oxygen to cells tissues or organs or a state of below normal oxygen levels.

Ice nucleation

the point at which the formation of ice begins in a given solution.


is a restriction in blood supply to tissues, causing a shortage of oxygen and glucose needed for cellular metabolism

Latent heat of fusion

the energy released, usually observed as heat, when water changes to ice in a biological system

Liquid crystalline phase

the state of a lipid bilayer or membrane in which the membrane is fluidic; lipids can diffuse freely inside the dual layer of the membrane. This usually occurs above the transition temperature, Tm.

Oncotic force

also called colloidal osmotic pressure, it is a form of osmotic pressure exerted by proteins or in the case of cryopreservation macromolecules in the solution, which may pull water from cells and have protective effects on cell membranes.

Osmotic equilibrium

is used to indicate that the concentration of a solute in water is the same on both sides of a semi-permeable membrane. While the water still passes through the membrane, there is no gain or loss on either side.

Osmotic pressure

the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a semi-permeable membrane


when very small (microscopic) ice crystals that may have formed during freezing are given the opportunity to grow during the re-warming of a sample.

Sample agitation

the process of gently mixing a sample during thawing to reduce/eliminate the formation of step thermal gradients within a sample which can be highly lethal.


also known as under-cooling; is the process of lowering the temperature of a liquid or a gas below its freezing point without it becoming a solid.

Thermal gradient

the rate of temperature change with through a body or across a surface.


glass transition temperature; the temperature region at which a substance undergoes a phase transition from a liquidous to a glassy state.


also known as the transition temperature of a lipid bilayer; it is the temperature at which a membrane shifts from being in a liquid crystalline phase to a gel phase.

Vehicle solution/carrier medium

a base solution in which a cryoprotectant is added and is usually designed to help facilitate cell maintenance under adverse conditions such as cooling below physiological temperature or freezing.

Viability assessment tiers

the strategy of utilizing multiple assays prior to or following cryopreservation to more effectively determine sample quality (viability and function).

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Baust, J.M., Campbell, L.H. & Harbell, J.W. Best practices for cryopreserving, thawing, recovering, and assessing cells. In Vitro Cell.Dev.Biol.-Animal 53, 855–871 (2017).

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