Cryopreservation of Cells Step by Step

Cryopreservation is crucial to the long-term preservation of cells in the lab, so it is important to understand the process of cell freezing.

Even the most diligent and dedicated scientist do not have the ability to maintain a cell line day after day for years. In addition, over time in culture, cells may undergo genetic changes, senesce, or become contaminated. Freezing can protect cells from these processes.

Freezing cells can be easily accomplished with only a few reagents, and storing cells in liquid N2 ensures a reliable, long-term source of cells.

Here, we'll take you through the ins and outs of freezing cells.

Select the Cells

Healthy, actively growing cells should be used for cryopreservation. It is best to passage the cells 1-2 days prior to freezing to ensure that they are in a log rhythmic phase of growth at the time of freezing. It is recommended to change the growth media 24 hours before freezing if the cells have not been passaged by then.

Check your cultures carefully for signs of microbial contamination. Growing cultures for several passages in antibiotic-free media before detection can make contaminants that might have gone undetected reach a more detectable level.

Harvest the Cells

Harvest cells using appropriate procedures for the cell type and be as gentle as possible. Once you have harvested the cells, wash off or inactivate any dissociating agents that may damage the cells.

Resuspend the Cells in Freezing Media and Aliquot to Cryovials

Freezing cells can be fatal. To avoid damage caused by, for example, ice crystal formation, osmotic stress, or membrane damage, a cryoprotectant is used to lower the freezing point of cells.

DMSO, as a 10% stock solution, is the most commonly used cryoprotectant (Wear gloves when working with DMSO as it easily penetrates the skin.). The most common freezing media is 90% FBS/10% DMSO. For less finicky cells and for tissue culture on a budget, 10% DMSO in cell growth media can also be used.

Make sure you have cryovials designed for liquid N2 storage. Vials should be labeled with a lab marker that will withstand liquid N2 and alcohol.

Cool the Cells

To allow water to drain out of the cells before freezing, freeze cells slowly. This is done using a cell freezing chamber.

The rate of cooling 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 decrease between 1°C and 3°C per minute. Larger or less permeable cells may need slower cooling rates because they take longer to dehydrate.

Some labs use programmable electronic cooling units that can precisely control the freezing process and yield uniform, reproducible results. Others use mechanical units that offer adequate control of the process at a less expensive price. One of the most economical and common methods for cell lines is to use an ultra-low temperature freezer with an insulated polystyrene foam box.

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. Most cell culture labs use liquid nitrogen tank.

Creative Bioarray Relevant Recommendations

Explore a variety of primary cultures, cell lines, immortalized cells and stem cells for your cell culture.

For research use only. Not for any other purpose.