Cryopreservation of Cells Guide | Corning

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Cell cryopreservation puts your cell lines in suspended animation, effectively stopping biological time. While it might seem the stuff of science fiction, cryopreservation is a fundamental process in cell culturing, a meticulous process that requires the utmost precision and care.

So how does the cryopreservation of cells work? Let's break this multistep process down.

Step 1: Select the Cells

Optimal cryopreservation occurs when cells are in the best possible condition and near the end of the logarithmic growth phase. Carefully examine your cultures for signs of microbial contamination. Growing cultures for several passages in an antibiotic-free medium before testing can make contaminants that might have gone undetected reach a more detectable level.

Examine your samples under a microscope, then test them by direct culture for bacteria, yeasts, fungi, and mycoplasmas. Mycoplasmas present unique issues because they can sneak through tests; you'll need to test culture stocks again after they've been frozen.

Step 2: Harvest the Cells

Harvest the cells using the proper procedure for the cell type — and be as gentle as possible. Once you've harvested the cells, wash off or inactivate any dissociating agents, which can damage the cells. Use a centrifuge only if you must, and be gentle — only use a force hard enough to yield a soft pellet.

Pool the contents of the harvested culture vessels to ensure the uniformity of the final frozen stock. Dilute or concentrate the cell suspension as needed to achieve twice the desired concentration.

Keep the cells chilled to slow cell metabolism and to prevent clumping. Cells can be gassed with carbon dioxide when necessary to prevent alkaline pH shifts.

Step 3: Store the Cells

Selecting the right cryoprotective agents prevents — or at least minimizes — cells from damage during cryopreservation. Choosing the right storage vessel is also critical. Choosing the wrong one carries numerous risks, including injury, damage to vessels, and contamination or loss of frozen stock. Heat-sealable glass ampules and polypropylene screw-capped vials (internal or external thread) are the most commonly used vessels for cryogenic storage, but sealing issues with the former have researchers and industry professionals increasingly preferring the latter.

Step 4: Cool the Cells

The cooling rate must be uninterrupted, and it must be slow enough to afford the cells time to dehydrate but fast enough to prevent damage from dehydration. For most animal cell cultures, the ideal cooling rate is a steady drop between 1°C and 3°C per minute. Larger or less permeable cells might need to cool more slowly because they take longer to dehydrate.

Some labs use programmable electronic cooling units, which provide precise control of the freezing process and yield uniform, reproducible results. Others use mechanical units that offer sufficient control of the process at a less expensive price. One of the most economical and common methods for cell lines is the use of ultracold freezers with insulated polystyrene foam boxes.

Step 5: Store the Cells

Once the stock is frozen, you need to move fast. Use an insulated container filled with dry ice or liquid nitrogen to transfer the frozen stock to permanent storage. Speed is the key to avoid warming the vials and damaging the cells.

Most cell culture labs use liquid nitrogen freezers, but the most important feature in any permanent frozen storage location is that it can reliably maintain temperatures below -130°C. Even a temporary rise in temperature can damage the cells.

Step 6: Thaw the Cells

Cooling cells must be done gradually, but the opposite is true when thawing cells. Rapid thawing reduces the formation of damage-causing ice crystals within the cells as they rehydrate. Place your container in warm water and stir it gently until it's completely thawed. For most cell cultures, thawing for 60 to 90 seconds at 37°C achieves the best results.

Step 7: Let the Cells Recover

Remove the cryoprotective agents from the cells as quickly and gently as possible to avoid the damage that prolonged exposure to these agents can cause. How you remove the cryoprotective agents depends on the type of agent and the type of cells — cells that are sensitive to cryoprotective agents, for example, require gentle centrifugation to remove the agent. When glycerol is the cryoprotectant, the sudden addition of a large volume of fresh medium to the thawed cell suspension can damage or destroy the cells. To avoid this, take the cells through several stepwise dilutions with an equal volume of warm medium every 10 minutes before further processing to give the cells time to adjust.

In general, most cells recover normally if the cryoprotective agent is removed through a medium change within six to eight hours of thawing.

When successfully preserved, frozen cells need little maintenance and can be a lifeline if you lose cell cultures to contamination or accident. Frozen cell cultures are especially useful for long-term experiments, as their suspended animation ensures that biological variants are kept to a minimum.