1. The cells to be cryopreserved should be in a state of good growth (log phase) and high survival rate, with a density of about 80-90%.
2. Check whether the cells still retain their unique properties before freezing.
For example, hybridoma should be tested for antibody production one to two days before cryopreservation.
3. Pay attention to the quality of cryoprotectant.
DMSO should be reagent grade, sterile and colorless (filtered with 0.22 micron FGLP Telflon or directly purchased sterile products, such as Sigma D-2650), aliquoted in small volumes of 5-10 ml, stored at 4 oC in the dark. Do not defrost multiple times. Glycerol should also be of reagent grade and stored in the dark after autoclaving. Use within one year after opening, because it will be toxic to cells after long-term storage.
4. Cell concentration for cryopreservation:
(1) Normal human fibroblast: 1~3 x 106 cells/ml
(2) Hybridoma: 1~3 x 106 cells/ml, the cell concentration should not be too high, some hybridoma will die after thawing 24 hours due to the high freezing concentration.
(3) Adherent tumor lines: 5~7 x 106, depending on the type of cell. Adenocarcinoma requires a higher concentration after thawing, while HeLa only needs 1-3 x 106 cells/ml.
(4) other suspensions: 5~10 x 106 cells/ml, human lymphocyte must be at least 5 x 106 cells/ml.
5. The cryoprotectant concentration is 5 or 10% DMSO. If the freezing conditions of the cells are uncertain, a backup culture should be used while cryopreserving to prevent freezing failure.
6. Freezing method:
(1) Traditional method: 4 oC 10 minutes ---> -20 oC 30 minutes ---> -80 oC 16-18 hours (or overnight) ---> Long-term storage in the vapor phase of liquid nitrogen tank.
(2) Program cooling: Use a constant-speed cooling machine to reduce from room temperature to –120 oC at a rate of –1 ~ -3 oC/min, and store it in the vapor phase of a liquid nitrogen tank for long-term storage. It is suitable for the preservation of suspension cells and hybridoma.
7. Material:
(1) Cultured cells that grow well
(2) Fresh medium
(3) DMSO (Sigma D-2650)
(4) Sterile plastic cryopreservation tube (Nalgene 5000-0020)
(5) 0.4% w/v trypan blue (GibcoBRL 15250-061)
(6) Hemocytometer plate and cover glass
(7) Constant velocity cooling machine (KRYO 10 Series II)
8. Steps:
(1) Change half or full amount of medium one day before freezing, and observe cell growth.
(2) Preparation of cryopreservation solution (preparation before use): Add DMSO to fresh medium, the final concentration is 5-10%, mix well, and put it at room temperature for later use.
(3) According to the operation of cell subculture, collect the cultured cells and take a small amount of cell suspension (about 0.1 ml) to count the cell concentration and survival rate before freezing.
(4) Centrifuge, remove the supernatant, add an appropriate amount of cryopreservation solution to make the cell concentration 1-5 x 106 cells/ml, mix well, and dispense into the labeled cryopreservation tube, 1 ml/vial, and Take a small amount of cell suspension for contamination detection.
(5) Cryopreservation method 1: The cryotube is placed at 4 oC for 10 minutes → -20 oC for 30 minutes → -80 oC for 16 to 18 hours (or overnight) → liquid nitrogen tank vapor phase for long-term storage.
(6) Freezing storage method 2: The freezing tube is placed in a constant velocity cooling machine with a set program, and then placed in a liquid nitrogen tank.
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