Cryopreservation of Cells Guide | Corning

Cell cryopreservation suspends cell line animation, effectively stopping biological time. This may seem like science fiction, but cell cryopreservation is fundamental to cell culturing. It's a meticulous process that requires exquisite precision and care.

Cryopreservation of Cells Step by Step Guide

The cryopreservation of cells requires careful attention. But how do you prepare cells for cryopreservation successfully? Follow these steps:

For optimal cryopreservation, cells must be in prime condition and near the end of the logarithmic growth phase. Make sure they have more than 80 percent confluency before freezing and carefully examine cultures for microbial contamination. Using an antibiotic-free medium to grow cultures for several pre-testing passages may foster enough growth to identify otherwise undetectable contaminants.

Examine samples under a microscope, and conduct a direct culture to test for bacteria, fungi, mycoplasmas, and yeasts. Mycoplasmas present unique challenges because they can pass undetected, so make sure to culture stocks again after freezing.

Step 2: Harvest the Cells

Be gentle when harvesting and vary your procedures by cell type. Strive for the optimal freezing concentrations. Low concentrations decrease viability; high concentrations can lead to cell clumping.

Wash off or inactivate any dissociating agents after harvesting because they can damage cells. If you use a centrifuge, only use 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 the concentration of the cell suspension as needed to achieve twice the desired concentration.

Keep cells chilled to slow cell metabolism and prevent clumping. Gas cells with carbon dioxide as necessary to prevent alkaline pH shifts.

Select the right cryoprotective agents, like dimethyl sulfoxide or glycerol, to minimize cell damage. Your storage vessel choice is also critical because improper containers present risks, including injury, vessel damage, contamination, or loss of frozen stock.

The most used cryogenic storage vessels are single-use storage containers, like Corning^® cryopreservation bags, and polypropylene screw-capped vials, like Corning internal or external thread cryogenic vials.

Step 4: Cool the Cells

Keep your cooling rate steady to ensure cell viability and integrity. It should be slow enough to allow for dehydration but fast enough to prevent dehydration-related damage. For most animal cell cultures, the ideal cooling rate is a steady drop between 1°C and 3°C per minute until they reach -80°C. Larger or less permeable cells might need to cool slower because they dehydrate slower.

Some labs use programmable electronic cooling units to precisely control the freezing process and yield uniform, reproducible results. Other labs use mechanical units that offer sufficient, more affordable process control. Consider using Corning Cool Cell^® alcohol-free cell-freezing containers to ensure cells freeze at the ideal cooling rate.

Once cells freeze, it's important to move quickly. Use an insulated container filled with dry ice or liquid nitrogen to transfer the frozen stock to permanent storage. Speed is critical to avoid cell warming or damage.

Most cell culture labs use liquid nitrogen freezers to maintain a consistent temperature below -140°C. Even a brief, temporary temperature rise can damage cells.

Step 6: Thaw the Cells

While you cool cells gradually, you thaw them rapidly. Quick thawing reduces the formation of damage-causing ice crystals inside cells during rehydration.

Put your container in warm water and stir gently until small ice pellets remain. Cells will continue to thaw as you spray them with ethanol and place them in the hood. You will get the best results by thawing cell cultures for 60 to 90 seconds at 37°C.

To avoid damage from prolonged exposure, remove cells from cryoprotective agents quickly and gently. Remove them based on the type of agent and cells. For example, gentle centrifugation can remove agents from cells sensitive to cryoprotective agents.

Be careful when glycerol is used as a cryoprotectant. Do not suddenly add a large volume of fresh medium to thawed cells — this can damage or destroy them. Instead, take the cells through several stepwise dilutions with an equal volume of warm medium every 10 minutes and let them adjust before further processing.

Most cells recover normally if you remove cryoprotective agents through a medium change within 6 to 24 hours of thawing.

Successfully preserved frozen cells need little maintenance. They can be a lifeline if you lose cell cultures to contamination or accidents. Frozen cell cultures can be useful for long-term experiments because their suspended animation ensures limited biological variants.