Why Freezing Bags Break

Description of the Causes for: The Failure of Cryopreservation Bags To Breakage

Author: Herb Cullis 4-30-09                

The reason for freezing bag failure has not been widely recognized. Bags fail because of carbon dioxide. The products frozen are living cells that have been maintained in nutrient rich tissue culture media prior to freezing. The cells metabolize sugars and produce carbon dioxide, which is initially carbonic acid or completely dissolved carbon dioxide. As the culture of cells awaits freezing it is usually taken from an incubator or room temperature and cooled in a refrigerator or on ice in preparation for the addition of DMSO or other cryoprotectant. During this 'cold incubation' production of carbon dioxide continues, and at lower temperatures the media has greater solubility for carbon dioxide so the solution accumulates large amounts of dissolved carbon dioxide. Then the cryoprotectant is added to the cold solution. The reason for doing this cold is that the DMSO dilution has a very large exotherm and the osmolality goes above 2000 milliosmoles. And then the solution is frozen. At the freezing point of the hyperosmotic solution (about -18C) ice freezes and the carbon dioxide is liberated as a gas, now trapped as small bubbles. As the temperature drops below -76C the carbon dioxide solidifies and decreases 960 volumes creating small vacuoles that soon become filled with oxygen and nitrogen. But when the temperature goes above -76C during thawing, the dry ice becomes a gas again and expands suddenly. People who are familiar with the process frequently/usually hear the ice cracking as the bag warms. The EVA and PVC bags are still brittle at -76C and cannot expand without cracking. FEP is still flexible and it just flexes. In one citing, some centers reported no failures and other reported many failures. The reason turned out to depend on the type of tissue culture media used (whether cells were metabolizing rapidly or not) and the technique for cryoprotectant addition (rapid cool down to ice temperature or gradual cool down). If a high quality media is used cells grow vigorously and CO2 is produced; the viability of the thawed product is better, but more of the bags are subject to break. If the cooling is performed in a fast process, there is less time to produce carbon dioxide within the sealed bag and there is a more likely chance the bags will survive. This is the science behind the story of how bags break during thawing.