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.
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
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
of Medical Sciences.