Vitrification-based Cryopreservation: An Ultra-rapid Cooling Technique to Preserve Biological Specimens at Very Low Temperatures for Long-term Storage

Vitrification, an ultra-rapid cryopreservation technique, allows living cells and tissues to be cooled and stored at extremely low temperatures for very long durations.

To vitrify a freshly harvested tumor tissue, first, take the tissue on a culture plate. Excise any necrotic regions, fat-containing regions, or blood clots from the tumor. Wash the tissue to remove any traces of blood.

Cut the tissue into slices of appropriate thickness. Now, transfer the slices into a tube containing a vitrification solution constituting DMSO, or dimethyl sulfoxide, and sucrose. These constituents act as cryoprotectants to protect cells against freezing damage.

Incubate at a low temperature. DMSO, a cell-permeating cryoprotectant, diffuses across the cell membrane and replaces water in the cells to prevent intracellular ice crystal formation. Concurrently, sucrose, a non-permeating cryoprotectant, acts extracellularly by facilitating cell dehydration and reducing intracellular ice crystal formation to mitigate the risk of cell death.

Place the equilibrated tumor slices onto a tissue holder. Next, transfer this assembly into liquid nitrogen to freeze at minus 196 degrees Celsius. This rapid cooling results in sample vitrification that solidifies the tissue into a glassy, amorphous state, while the high concentration of cryoprotectants prevents ice crystal formation and cell damage.

The vitrified tissue remains in a state of suspended metabolic activity.