Cell Freezing Media and Salt-Based Solutions

Cells lose water during freezing – they shrink in size and quickly lose viability if they go below a minimum volume. Adding cryoprotectant agents like dimethylsulfoxide (DMSO) mitigates these effects.

The medium should include an agent that can absorb water to prevent ice crystal formation during the slow freezing process and maintain cell viability.

Serum is a black box that contains components that are not necessary for cryopreservation, and might even harm cells during freezing and thawing. Animal component-free solutions help enhance viability and recovery, in addition to reproducibility and consistency.

DMSO is cytotoxic to cells. During cryopreservation, cells can only stay in DMSO diluted in solution to 10% and under.

You can freeze cells without DMSO as long as you have nontoxic protective agents with the same effect.

Most cell cultures can be stored for many years, if not indefinitely, at temperatures below –130°C. ATCC has recovered cells from cultures cryopreserved for more than 40 years.  The advantages of cryopreservation far outweigh the required investment in equipment and reagents. These advantages include:

• Generating safety stocks to prevent losing a culture from equipment failures or contamination by microorganisms or other cell lines.
• Saving time, energy, and materials that would be required to maintain cultures not in use.
• Preserving cells with finite population doublings that will ultimately senesce.
• Preventing phenotypic drift arising from genetic instability and/or selective pressure.
• Creating a standard reagent that can be used for a series of experiments (source: ATCC guide for Animal Cell Culture).

1. Centrifuge cells at 300 – 400 × g for 4 – 5 minutes to obtain a cell pellet. Then aseptically remove the supernatant.                                                                                        
2. Suspend the pellet in cryopreservation solution at 2°C – 8°C. Mix thoroughly and transfer the suspension to a cryovial (e.g., 1.0 mL of suspension in a 1.5 mL cryovial).        
3. Freeze the cells gradually (1°C – 2°C per minute) using a controlled rate freezing system and store the vials in liquid nitrogen (vapor phase). Alternately, place the vials in a freezing container and transfer to -80°C overnight. Transfer the cryovials into liquid nitrogen the following day – we recommend the vapor phase.                     
4. Determine the efficiency of cryopreservation by thawing one vial after 24 hours of storage in liquid nitrogen.