Page 10 - Cell Culture Training Hand Book - A4 Edited 20.1.2020

CRYO PRESERVATION

Freezing Media recipe (1 ml)

Content
No. Items Concentration Volume (µl)

1 Dimethyl sulfoxide (DMSO) 10% 100

2 Culture media 90% 900
Total volume: 1000

Reagents
+2
1. DPBS 1x (Dulbecco’s Phosphate Buffered Saline) (- Ca - Mg )
+2
2. Trypsin EDTA 0.25%
3. Culture media (DMEM with 10% FBS)

4. Freezing media
5. Trypan Blue

Protocol
1. Remove culture media from the flask.
2. Wash cells culture with 5ml of DPBS (- Ca - Mg ) for 2 times
+2
+2
3. Add 2 ml of trypsin to detach the cells.
4. Incubate the culture for 5 minutes at at 37°C.
5. Tap the flask gently, then observe the culture under microscope to ensure cell detachment.
6. Add 4 ml of culture media to neutralise trypsin.
7. Transfer the cell suspension into fresh 15 ml falcon tube.
8. Take 10 µl of the cell suspension and mix with 10µl of Trypan Blue to determine the total
cells number.
9. Centrifuge the remaining cell suspension in the falcon tube at 1,500 rpm for 5 minutes.
10. Remove the supernatant. Tap tube gently to loosen pellet and resuspend the pellet (at least
50 x, and avoid bubbles) with 5 ml of freezing media.

11. Transfer 1 x 10 cells/ml of mixture into cryo vials.
6
12. Seal the vials with parafilm.
13. Transfer all vials into Mr. Frosty and place it into -20°C freezer, follow with -80°C freezer, and

finally into the nitrogen tank .

Tips
1. It is a cryoprotective agent. DMSO is known to facilitate the entry of organic molecules into tissues. Handle reagents containing
DMSO using equipment and practices appropriate for the hazards posed by such materials.
2. Complete culture media in the presence of a cryoprotective agent such as dimethylsulfoxide (DMSO).
6
3. Depending on your cell type, it is a common practise to aliquot 1 ml of cells (1 x 10 cells) into every vial.
4. Cryoprotective agents reduce the freezing point of the culture media. Slower cooling rate will greatly reduce the risk of ice
crystal formation, which can damage cells and cause cell death.

Cell Culture Training Hand Book | CMBL Page 10 of 12