Work Instruction
WI0014
Preparation of Cells for Liquid Nitrogen Storage
Rev: C
1. Materials
1.1. Culture Media, varies with cell type
1.2. Fetal Bovine Serum (FBS)
1.2.1. Manufacturer: Invitrogen
1.2.2. Catalog No.: 16000
1.3. Dimethylsulfoxide (DMSO), Sterile
1.4. Cryo Freeze Container (contains isopropyl alcohol)
1.4.1. Manufacturer: Nalgene
1.4.2. Catalog No.: 5100
1.5. 2.0 mL Sterile Cryogenic Vials with Internally Threaded Screw-Caps
1.5.1. Manufacturer: Corning
1.5.2. Catalog No.: 430488
2. Safety
2.1. Be careful with the screw-cap type tubes when working with liquid nitrogen. If
liquefied gas enters tube, the tube can explode loudly enough to cause hearing
damage and also very forcibly if the tube warms sufficiently. Keep the tubes
cold and make sure no liquid nitrogen enters, especially if you need to re-tighten
the cap. The internal threads may reduce risk of the cap lossening.
2.2. If you must remove a box of vials from the liquid nitrogen container for an
extended period of time, place some liquid nitrogen in a small styrofoam
container and dip the box in while working. This will reduce the risk of the
containers warming.
2.3. Wear thermally protective gloves whenever possible to reduce the risk of
frostbite. Don’t wear mesh or cotton fiber gloves since liquid nitrogen could get
through.
3. Procedure
3.1. The day before the freeze, split the cells so they are in log phase when you freeze
them. Based on your knowledge of the growth rate, it should be possible to
optimize the split so you have a good density to freeze the next day but still have
not exited log phase.
3.2. If the cells are adhered to the culture surface, detach them.
3.3. Determine the concentration of cells in solution with the hemacytometer.
3.4. Aliquot 5 × 106 – 1 × 107 cells per centrifuge tube.
3.5. Centrifuge cells, usually 300×g for 3 minutes.
3.6. Prepare cell freeze media:
3.6.1. 70% cell culture media (depends on your cell type)
3.6.2. 20% FBS
3.6.3. 10% sterile DMSO
3.7. Remove the supernatant from the centrifuge tube and add in 1.0-1.5 mL cell
freeze media.
3.8. Quickly transfer cells to the 2.0 mL sterile screw-cap tube.
3.9. Quickly transfer the cells to the cryo freeze container, which controls the rate of
cooling. The container should be at room temperature to start.
3.10.
Quickly transfer the isopropyl alcohol bath to the -80ºC freezer.
bme.virginia.edu/lawrence
Date: 5/05/08
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Work Instruction
WI0014
Preparation of Cells for Liquid Nitrogen Storage
Rev: C
3.11.
Incubate for 8 hours to overnight. Cells may be stored at -80ºC for up to a
week if necessary.
3.12.
Transfer the cells to the liquid nitrogen storage.
3.13.
Update the lab liquid nitrogen content inventory on the UVa collab web
site (https://collab.itc.virginia.edu/).
4. References
4.1. www.ucsf.edu/desailab/Support/Procedure%20for%20freezing%20down%20cell
s.doc
5. Revision History
Date
Rev Description
5/05/08 C
Revised optimal number of cells to freeze
2/24/08 B
Added detail to the safety section and
details to split the cells the day before
freezing. Revised document name.
1/29/08 A
Added detail to the tube description and a
safety section.
12/30/07 Initial release.
bme.virginia.edu/lawrence
Date: 5/05/08
Revision Author
Brian J. Schmidt
Brian J. Schmidt
Brian J. Schmidt
Brian J. Schmidt
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