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

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.