CoolCell® Containers Bolster Patent Application for Novel Cell Freezing Method

Patent Office relief on the Herbert C. Hoover Building in Washington, DC. Photo by Neutrality. From Wikimedia.

A surprisingly simple innovation may substantially increase viability of cells after freezing. The creators of a novel cell freezing method file for a patent in the US and use BioCision’s CoolCell® containers in their protocols.

Manufacture of cell therapy products is most often performed both physically and temporally disconnected from the final delivery to a patient in the clinic. Cell products must therefore usually be frozen after production. However, the cold truth is that not all cells survive the cell freezing procedure – sometimes as many as one third of all cells die in the process.

Researchers are trying many different lines of attack to increase cell survival, functionality and viability after cell freezing. They search for less toxic and more cryoprotective substances compared to the standard DMSO (we recently blogged about encouraging alternative cryoprotectants, such as the silkworm-derived protein sericin, buffalo ocular fluid, synthetic betaine-like molecules or the chemical element boron). They investigate many different temperatures and speeds to freeze and store cells. They seek better ways to transport cells to avoid temperature fluctuations that may reduce subsequent cell viability.

Now, a team of scientist from Spain applied for a US patent to add another innovation to the process. 1 Essentially, the researchers found that freezing the cells in a more concentrated form boosts cell survival. They therefore decided to modify the regular cell freezing method by addition of an easy step. First, they incubated cells with standard cryopreservation media long enough to let the media components enter the cells’ interior and replace the cellular water content. Then, instead of freezing the cell suspension directly, they separated the excess medium from the cells by simple centrifugation. Once separated, the pelleted cells were frozen by controlled-rate cell freezing, using BioCision’s CoolCell® freezing containers to ensure a consistent cell freezing technique that proceeded at the recommended rate of -1°C / min.

As a neat added novelty, the scientists then devised a two-chambered container which includes an access canal between the two compartments. They used this vessel to separately freeze the cell pellet and a non-toxic cell growth medium. Once the container was subsequently removed from cryostorage for thawing, gravity ensured that the medium slowly dripped through the access canal into the cell population as it thawed. The effect was a minimized osmotic shock for the precious cells, and a diminished risk of contamination, during the thawing procedure.

Essentially, any two-chambered vessel with access between the two compartments would work, but the researchers determined that a container that results in a surface/volume ratio between 8 and 21 cm-1 gives the best results for most cell types. Why? Turns out that this ratio results in the most homogeneous cooling and thawing rate possible across the entire sample.

In their patent application, the researchers were able to show much improved cell viability after cell freezing, with increases of up to 30%. This held true for a variety of eukaryotic cells, including human mesenchymal stem cells, mouse embryonic stem cells and human embryonic kidney cells. An added benefit of the novel procedure is the lower toxicity of the thawed cell suspension for many downstream applications as excess cryopreservation medium, and therefore excess toxic cryoprotectants such as DMSO, are removed in the process. These benefits, together with the simplicity of the innovation, may reward the researchers with recognition, praise, and, yes, well-deserved cold hard cash.


[1] Hernan Izquierdo, R. et al. Method of freezing cells. United States Patent Application 20150140656. Published on 05/21/2015., accessed Nov 2, 2015.