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.

After euthanizing the mice, use sterile forceps and scissors to slowly isolate the tumor from the mice. Wash the tumor tissues in DPBS and a 10-centimeter dish. Use forceps and scissors to dissect and remove necrotic areas, fatty tissue, blood clots, and connective tissue.

Next, use a mold to cut the tumor tissues to a maximum thickness of 1 millimeter. Wash the tumor slices with DPBS in a 10-centimeter dish. To begin the vitrification process, use forceps to transfer the slices into tube V1 and incubate at 4 degrees Celsius for 4 minutes. Roll and invert the tube briefly and incubate at 4 degrees Celsius for another 4 minutes.

Next, pour the V1 solution and slices into a 10-centimeter dish and use forceps to transfer the slices into tube V2. Incubate at 4 degrees Celsius for 4 minutes. Roll and invert the tube briefly and incubate at 4 degrees Celsius for another 4 minutes. After this, pour the V2 solution and the slices into a 10-centimeter dish.

Transfer the slices into tube V3 and incubate at 4 degrees Celsius for 5 minutes. Roll and invert the tube briefly and incubate at 4 degrees Celsius for another 5 minutes.

Make sure that all of the slices sink to the bottom of the tube. If several slices remain floating, roll and invert the tube briefly and incubate at 4 degrees Celsius until all of the slices sink completely. Then, pour the V3 solution and slices into a 10-centimeter dish. Cut the tissue holders to the proper length and place them on sterile gauze. Transfer the slices onto the holders. Wrap the holders with gauze.

Using forceps, place the holders in liquid nitrogen and let them incubate for 5 minutes. After this, label the cryogenic vials with the tissue information. Transfer the holders with tissue slices into the vials, which will be stored in liquid nitrogen.