Why do ice crystals kill cells during cryopreservation?

Cryopreservation is a great way to store cells for a prolonged period of time. For example, preserving bacterial cultures kept at -80 ºC can ensure they remain viable for over five years. The best way to preserve samples for long-term use involves the use of an ultralow temperature (ULT) freezer.

Why do ice crystals kill cells during cryopreservation?

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However, some challenges can arise when freezing samples at such low temperatures. Water expands when it freezes, which leads to ice crystal formation. This expansion and crystal formation can cause fatal damage to cells, consequently rendering them dead.

For this reason, before subjecting cells to ultralow temperatures, they must be protected from the crystallization of water.

Ice crystals kill cells

Ice crystals regularly occur when water freezes. At low temperatures, water molecules arrange themselves in an orderly fashion, forming crystals. These crystals can have pointed ends and sharp edges, puncturing cell walls and membranes and causing structural damage and death.

Ice crystal formation can also dehydrate cells. Extracellular ice forms much faster than intracellular ice. Resultingly, the concentration of solutes is increased on the outside of the cells, as there is more liquid water inside the cells.

Osmosis then pushes intracellular water out, leading the cells to shrivel up and dry out. Cell death follows through dehydration and solute toxicity, as the concentration of solutes inside the cells becomes too high for them to tolerate.


To ensure cryopreservation works properly, preventing ice crystals from forming is crucial. A frequently applied method involves the use of cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol. These substances hinder the formation of ice crystals when samples are placed in ULT freezers.

Mechanical damage and dehydration to the cells are also avoided as cryoprotectants prevent the formation of ice crystals both inside and outside cells. Different samples require different types of cryoprotectants. For bacterial cultures, glycerol is commonly used. For cell cultures, DMSO is the recommended cryoprotectant.

Reviving cells

When it comes to reviving cryopreserved cells, they need to be returned to biologically functioning states. Protocols may vary depending on what cryoprotectant is used during cryopreservation.

For instance, when reviving bacteria frozen with a glycerol stock, it is essential that thawing does not occur at any stage in the storage process. Thawing can damage bacterial cells, preventing them from growing and proliferating in culture.

When DMSO is used, it is necessary to heat the cultures in a 37 ºC water bath for a few minutes until thawed. Then, a fresh culture medium should be applied to wash the cells to flush out the DMSO. If the cryoprotectant is not removed quickly enough, it may cause damage to the cells.

Maintaining cell viability

It is absolutely necessary to ensure cryopreserved samples are kept at a consistent temperature in order for them to remain viable for years. Repeated freeze-thaw cycles can lead to cell deterioration.

To prevent this, cryopreserved stocks must not be removed from a ULT freezer for prolonged periods of time. It is also recommended to keep freezer doors open for as little time as possible – just a few seconds. This avoids any sizable temperature fluctuations that can be damaging to cryopreserved samples.

About inTEST Thermal Solutions

inTEST Thermal Solutions (iTS) specializes in the design and manufacture of precision temperature control systems. Our breadth of products and in-house engineering capabilities allow us to be a single-partner-solution for thermal test, process cooling, and cold storage applications. We are recognized globally for our expertise in precise temperature control and simulation of extreme thermal environments, from -185 to +500 °C, with rapid transitions or long dwell times. The iTS family includes four product brands: Temptronic, Sigma Systems, Thermonics, and North Sciences. inTEST Thermal Solutions is a wholly owned subsidiary of inTEST Corp, Mount Laurel, New Jersey, USA.

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Last updated: Mar 3, 2023 at 5:50 AM


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