How to Cryopreserve Mammalian Cell Lines?

Cryopreservation is a method of preserving cells by freezing them at very low temperatures, usually in liquid nitrogen. Cryopreservation allows cells to be stored for long periods of time without losing their viability or functionality. Cryopreservation is useful for maintaining cell lines, avoiding contamination, minimizing genetic drift, and saving space and resources.

In this blog post, I will describe the basic principles and procedures of cryopreservation of mammalian cell lines, and provide some tips and references for further reading.

Principles of cryopreservation of mammalian cell lines

The basic principle of successful cryopreservation and resuscitation is a slow freeze and quick thaw. This is because rapid freezing can cause ice crystals to form inside and outside the cells, which can damage the cell membrane and organelles. On the other hand, slow freezing can allow the cells to dehydrate and shrink, reducing the intracellular ice formation. Moreover, slow freezing can also allow the cells to adapt to the low temperature and osmotic stress by activating protective mechanisms, such as the expression of heat shock proteins or the synthesis of cryoprotectants.

To achieve a slow freeze, a cryoprotective agent (CPA) is usually added to the freezing medium. A CPA is a substance that can lower the freezing point of water and prevent ice crystal formation. The most commonly used CPA for mammalian cell lines is dimethyl sulfoxide (DMSO), which can penetrate the cell membrane and protect the intracellular structures. Other CPAs that can be used are glycerol, ethylene glycol, propylene glycol, or trehalose. The concentration of CPA should be optimized for each cell type, as too high or too low concentrations can be toxic or ineffective.

The freezing medium also contains a base medium and a protein source. The base medium provides the nutrients and buffering capacity for the cells. The protein source provides additional protection and stabilization for the cells. The base medium and protein source can be either serum-containing or serum-free, depending on the culture conditions of the cell line. Serum-containing medium has the advantage of providing a variety of proteins, such as albumin, globulins, and growth factors, that can enhance cell viability and recovery. However, serum-containing medium also has the disadvantage of introducing variability and contamination to the culture system. Serum-free medium has the advantage of providing more control and consistency over the culture conditions. However, serum-free medium also has the disadvantage of requiring more optimization and supplementation for each cell type.

The optimal freezing rate for mammalian cell lines is about 1°C to 3°C per minute. This can be achieved by using a controlled-rate freezer or an isopropanol chamber that can regulate the temperature gradient. Alternatively, an insulated box or a CoolCell® device can be used to place the cryovials in a -80°C freezer and achieve a similar freezing rate.

After reaching -80°C, the cryovials should be transferred to liquid nitrogen storage at -196°C or below. The cells should be stored in the gas phase above the liquid phase to avoid cross-contamination or explosion due to liquid nitrogen leakage.

Procedures of cryopreservation of mammalian cell lines

The following procedures are based on the protocols from Thermo Fisher Scientific and Abcam. You may need to adjust the procedures according to your specific cell type and culture conditions.

Freezing adherent cell lines

1. Label cryovials with the date, name of researcher, cell number, passage number, cell type, and any other relevant information.
2. Prepare freezing medium by mixing 90% fetal bovine serum (FBS) or conditioned medium with 10% DMSO or glycerol. Warm the freezing medium to 37°C before use.
3. Detach cells from the culture vessel by removing the culture medium, washing with phosphate-buffered saline (PBS), and adding enough trypsin or another suitable enzyme. Incubate for about 2 minutes in a 37°C incubator until the cells are detached.
4. Resuspend cells in complete growth medium and transfer into a centrifuge tube.
5. Count cells using a hemocytometer or a cell counter to determine their viability and concentration. The viability should be at least 75% for successful cryopreservation.
6. Centrifuge cells at 200 x g for 5 minutes and discard the supernatant.
7. Resuspend cells in freezing medium at a concentration of about 5 x 10⁶ to 1 x 10⁷ cells/mL.
8. Aliquot 1 mL of cell suspension into each cryovial and secure the lids tightly.
9. Place cryovials in a controlled-rate freezer or an isopropanol chamber and freeze them at a rate of 1°C to 3°C per minute until they reach -80°C.
10. Transfer frozen cryovials to liquid nitrogen storage and store them in the gas phase above the liquid.

Freezing suspension cell lines

1. Label cryovials with the date, name of researcher, cell number, passage number, cell type, and any other relevant information.
2. Prepare freezing medium by mixing 90% FBS or conditioned medium with 10% DMSO or glycerol. Warm the freezing medium to 37°C before use.
3. Transfer cells from the culture vessel into a centrifuge tube.
4. Count cells using a hemocytometer or a cell counter to determine their viability and concentration. The viability should be at least 75% for successful cryopreservation.
5. Centrifuge cells at 200 x g for 5 minutes and discard the supernatant.
6. Resuspend cells in freezing medium at a concentration of about 1 x 10⁷ to 5 x 10⁷ cells/mL.
7. Aliquot 1 mL of cell suspension into each cryovial and secure the lids tightly.
8. Place cryovials in a controlled-rate freezer or an isopropanol chamber and freeze them at a rate of 1°C to 3°C per minute until they reach -80°C.
9. Transfer frozen cryovials to liquid nitrogen storage and store them in the gas phase above the liquid.

Tips for cryopreservation of mammalian cell lines

Here are some tips to improve the efficiency and quality of cryopreservation of mammalian cell lines:

• Use fresh and healthy cells that are in an exponential growth phase and have not been overgrown or contaminated.
• Use the recommended freezing medium for your cell type and culture conditions.
• Avoid exposing the cells to room temperature or DMSO for longer than 10 minutes, as this can cause stress and damage to the cells.
• Label the cryovials clearly and keep a record of their location and contents in a database or spreadsheet.

Conclusion

Cryopreservation of mammalian cell lines is a useful technique for preserving cells for future use. By following the principles and procedures outlined in this blog post, you can freeze your cell lines efficiently and effectively.