How to freeze and defreeze mesenchymal stem cells in the Nitrogen tank

Materials and equipment

1. Culture medium, 2. Trypsin-EDTA, 3. DMSO with cell culture grade 4. FBS, 5. Cryovial, 6. Cell freezer chamber,  7. -80 Freezer, 9. Nitrogen tank.

Method of freezing cells:

1. Passage the cells 1 to 2 days before freezing to make sure they are growing in the rhythmic phase during freezing. If you do not passage, be sure to change the culture medium 24 hours before freezing.

2. The most common material for protecting cells from freezing (cryprotectant) is DMSO in stock solution (10%), i.e., FBS at 90% and DMSO at 10%.

3. Wear gloves when using DMSO, as it penetrates the skin easily.

4. Store cells in a volume of 1 ml per vial.

5. Be careful to use a cryovial designed to store in liquid nitrogen.

6. Use an alcohol-resistant lab marker and liquid nitrogen to write on the vials.

7. Write the passage number (P) and the number of cells (N) on the cryovials.

8. Use the isopropanol storage chambers for temporary storage of the vials in the freezer -80 and then close the lid and put in the -80 freezer for at least 4 hours. If you do not have a chamber containing isopropanol, you can use Styrofoam rack. Place the cryovials in one of the racks and cover and glue them with the other rack.

9. It is better to store in the -80 freezer overnight and then transfer to a nitrogen tank.

 

How to melt ells (Thawing Cells)

1. Unlike freezing, which should be done slowly, melting cells should be done quickly.

2. Preheat the culture medium to 37 ° C by placing it in a Ben Murray.

3. Remove the vials containing the cells from the nitrogen tank and place them in Ben Murray at 37 ° C until the ice is completely melted.

4. Pour the volume of one ml vial of frozen cells in 10 ml of heated culture medium ( No. 2)

5. Because the melted cells have a brittle membrane, do not try to remove the freeze solution immediately by centrifuging the resulting medium, so you should transfer the cells to a flask, dish or well, and change them after precipitation of the cells to the bottom of their container. Therefore, it is recommended that you do this as late as 24 hours.

 

Dr. Mansour Esmaeili Dahaj,

Associate Professor,

Department of Physiology,

Yazd University of Medical Sciences.