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

Overview

In this video, we demonstrate an ultra-rapid cryo vitrification method for preserving biological samples. The method entails the use of a combination of cryoprotectants to protect the biological tissue from freezing damage by preventing intracellular ice crystal formation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tissue Cryopreservation

NOTE: This part primarily references the methods for the Live Tissue Kit Cryo Kit. The main kits and equipment are listed in the Table of Materials.

1. Euthanize the mice with an IRB-approved method when the tumor is greater than 10 mm in diameter.
2. Using sterile forceps and scissors, slowly isolate the tumor from the mice.
3. Wash the tumor tissues with DPBS in a 10 cm dish. Dissect and remove necrotic areas, fatty tissue, blood clots, and connective tissue with forceps and scissors.
4. Cut the tumor tissues to a maximum of 1 mm thickness with a mold.
5. Wash the slices with DPBS in a 10 cm dish.
   NOTE: The vitrification process involves the use of tubes labeled V1/V2/V3 in steps 1.6-1.8. The main ingredients are DMSO and sucrose.
6. Transfer the slices into tube V1 with forceps and incubate the tube at 4 °C for 4 min. Roll and invert the tube briefly, and place it at 4 °C for another 4 min.
7. Pour the V1 solution and slices into a 10 cm dish. Transfer the slices into tube V2 with forceps, and incubate tube V2 at 4 °C for 4 min. Roll and invert the tube briefly, and place it at 4 °C for another 4 min.
8. Pour the V2 solution and slices into a 10 cm dish. Transfer the slices into tube V3 with forceps, and incubate the tube at 4 °C for 5 min. Roll and invert the tube briefly, and place it at 4 °C for at least 5 min. Make sure the slices all sink to the bottom of the tube.
   1. If several slices remain floating, roll and invert the tube briefly, and place the tube at 4 °C again until all slices sink completely; if necessary, discard the floating slices.
9. Pour the V3 solution and slices into a 10 cm dish.
10. Cut the tissue holders to the proper length and place them on sterile gauze. Transfer the slices onto the holders. Wrap the holders with the gauze and place them in liquid nitrogen using forceps, followed by incubation for 5 min.
11. Label the cryogenic vials with the tissue information.
12. Transfer the holders with tissue slices into cryogenic vials, which are stored in liquid nitrogen.

Materials

Name	Company	Catalog Number	Comments
Live Tissue Kit Cryo Kit	Celliver Biotechnology, Shanghai, China	LT2601
Tissue-processed molds and auxiliary blades	Celliver Biotechnology, Shanghai, China	LT2603
DPBS	Basalmedia Technology, Shanghai, China	L40601