3D Cell Cryopreservation Best Practices

Cryopreservation of cells and cell lines is a key technique in 3D cell culture. It preserves cells, tissues, organoids, and other biological constructs by cooling the cells to extremely cold temperatures. Maintaining cell viability through ice formation and thawing is critical.

Thanks to temperature control equipment and cryoprotective agents (CPAs), the success rate of the technique is higher than ever, according to a review published in Integrative Medicine Research.

How Does Cryopreservation Work?

Cryopreservation involves combining the cryoprotective agents (CPAs) with cells before cooling them, then freezing and storing the cells. If cells are frozen quickly, ice crystal formation, membrane damage, and osmotic shock will cause cell death. Thus, it is critical to freeze slowly. CPAs are used to affect the rate of water transport, ice crystal growth, and nucleation.

CPAs reduce the damage cells and tissues incur during the freezing and thawing process. Cells are stored in cryogenic storage containers, such as Corning® CoolCell® alcohol-free freezing containers. When the process is done correctly, the delicate structure of cells is preserved, and the cells can be thawed and remain viable.

Cryopreservation of 3D Cell Cultures

Cell cultures such as organoids comes with significant challenges. Long-term storage of organoids would allow for continued organoid technology development and clinical translation, but ice crystal formation makes freezing complex tissues difficult.


In current practice, cells, including organoids, are frozen in a medium with a cryoprotectant and high concentrations of serum, and the cooling temperature is lowered slowly — about 1°C each minute. While the protracted freezing process can prevent ice formation, crystal formation from devitrification is still a problem, according to a study published in Advanced Biosystems, and it can damage cells and disrupt cell-cell interactions.

The researchers conducting the study developed a scalable organoid culture and cryostorage system based on hydrogel capsules, surrounding a core of Corning® Matrigel® matrix with a shell of alginate. This system allowed for a better recovery rate, as the hydrogel layers, the scientists surmised, protected the organoids from mechanical damage during the freezing and thawing processes.

Gel Systems

A study in the International Journal of Molecular Science evaluated natural polymer hydrogel systems, synthetic polymer hydrogel systems, and supramolecular hydrogel systems as conduits for cryopreservation. Hydrogel systems offer excellent biocompatibility because of their unique 3D structures.

The natural hydrogel system encapsulated the initial cell with chitosan or alginate and minimized cell damage by containing ice crystal formation within the hydrogel structure. Synthetic polymer hydrogel worked well, but the authors hypothesize that its strong chemical bond might make removing cells after thawing difficult.

Practical Applications

In the Advanced Biosystems study, the core-shell capsule system enabled the scalable production of intestinal organoids and protected the organoids from mechanical damage during cryopreservation.

Long-term storage of organoids and other 3D cell cultures enables them to be readily available for distribution for research and clinical transplantation.