Cell cryopreservation

1. Programmed freezing steps (gradient cooling is required)

A. Prepare the freezing solution. The recommended ratio is 55% basic medium + 40% serum + 5% DMSO, or 90% FBS +10% DMSO. Our recommended product is Pricella General Freezing Medium (Ref. PB180436).

B. Count the total number of prepared cell suspension.

C. Centrifuge the cell suspension at 1200rpm (250g) for 3 minutes. After centrifugation, remove the supernatant as much as possible.

D. Add the configured freezing solution for re-suspension, and adjust the cell concentration to 3×106 ~ 1×107cells/mL.

E. Aliquot the cells into cryogenic vials, volume at 1~1.5ml per vial.

F. Transfer the vials into a cell freezing container, and then put the container directly into the refrigerator at -80℃ overnight.

G. The next day, transfer the cells from the -80℃ refrigerator and quickly put them in liquid nitrogen for long-term storage. When transferring, it is necessary to take cooling measures on the cryogenic vials and avoid direct exposure to normal temperature.

H. If there is no cell freezing container, put the cells at different temperatures in the following order: room temperature → 4℃ for 20min → -20℃ for 30min → -80℃ for overnight → liquid nitrogen storage.

Note:

I. Complete or special medium containing Penicillin-Streptomycin is not recommended for cell cryopreservation, as it will affect the cell survival rate. 

J. DMSO will release heat during preparation. So to avoid burning cells, it can only be used after cooling. 

K. After cell centrifugation, remove as much supernatant as possible to reduce the residual which may dilute the freezing solution. 

L. Manual gradient cooling is not recommended. If the temperature is unstable, it’s easy to reduce the cell survival rate. 

M. The amount of isopropyl alcohol in the cell freezing container must be higher than the lowest scale line. Isopropyl alcohol needs to be replaced once after 5 times of cryopreservation, so as not to affect the storage effect. The cell freezing container should be restored to room temperature before continuing to use, and can not be directly used after being taken out of the refrigerator.

N. After adding the freezing solution, the less time the cell suspension is placed at room temperature the better. DMSO will cause great damage to cells at room temperature. After aliquot, transfer the cells to the -80℃ refrigerator immediately. Do not need to put them at 4℃.

O. After overnight at -80℃, cells can be transferred to liquid nitrogen for long-term storage. During the process of transferring to liquid nitrogen, the container should be kept cold, and it should not be exposed at room temperature for a long time to avoid melting. 

P. If there is no liquid nitrogen tank, the cells must be placed near the inside when stored at -80℃, and the times of switching the refrigerator should be minimized, so as to avoid temperature instability.

Q. Regularly in a certain time, resuscitate a vial of cryopreserved cells, observe the cryopreservation effect, and test the survival rate of cells. At the same time, living cells should be well cultured. Theoretically, cells can be stored in liquid nitrogen for a long time. For safety, cells can be frozen in batches or resuscitated in different time periods after freezing, so as to ensure the vitality of frozen cells and avoid cell shortage caused by cell inactivation.

2. Non-programmed freezing steps (Serum-free freezing solution is required)

A. Warm the freezing solution to room temperature in advance. Our recommended product is Pricella Freezing Medium (Serum-free & animal origin-free) (Ref. PB180438). 

B. Count the total number of prepared cell suspension . 

C. Centrifuge the cell suspension at 1200rpm (250g) for 3 minutes. After centrifugation, remove the supernatant as much as possible.

D. Add Freezing Medium (Serum-free & animal origin-free) (Ref. PB180438) for re-suspension, and adjust the cell concentration to 3×106 ~ 1×107cells/mL.

E. Aliquot the cells into cryogenic vials, volume at 1~1.5ml per vial.  

F. Transfer the vials into the refrigerator at -80℃ overnight directly. The cell freezing container is not needed.

G. The next day, transfer the cells from the -80℃ refrigerator and quickly put them in liquid nitrogen for long-term storage. When transferring, it is necessary to take cooling measures on the cryogenic vials and avoid direct exposure to normal temperature.

Note: 

A. Since the cell freezing container is not used, it is necessary to take cooling measures on the cryogenic vials during the transfer to liquid nitrogen. It’s suggested to protect the vials with dry ice or a small amount of liquid nitrogen. Do not hold the vials directly by hand, and pay attention to operation safety.

B. The transfer process should be fast to avoid melting after exposure to room temperature, which may affect cell survival rate. 

C. If there is no liquid nitrogen tank, it must be placed near the inside when it is stored at -80 degrees, so as to avoid temperature instability caused by switching the refrigerator, and minimize the number and time of switching the refrigerator.