- Freezing medium can be used to preserve cell lines for long term at ultra-low temperature. This method of preserving cell line is called cryopreservation.
- An ideal freezing medium must allow 100% recovery without causing any change in cell characteristics after revival.
- Freezing medium is nothing but a complete medium supplemented with high concentration of serum and a cryoprotective agent such as DMSO or glycerol.
- As the name suggest, cryoprotective agents protect cells from lysis due to ice crystal formation at temperature below freezing point.
- Serum concentration as high as 90% can be used in freezing medium. High serum concentration improves cell viability and recovery after thawing.
- Serum-free chemically-defined freezing media are also available. They are very useful to preserve those cell lines which are maintained in serum-free chemically defined medium.
- Both serum-containing and serum-free freezing medium are available commercially.
- Serum-containing medium is used for cell lines which are maintained in serum-supplemented growth medium. We recommend to check cell line manual for optimal freezing medium.
- Fetal bovine serum (FBS) or Fetal calf serum (FCS)
- DMSO (sterile)
- Complete growth medium (optional)
- Equipment and disposables
- 50 ml sterile polypropylene tubes
- Laminar flow hood
- Use the same growth medium which is used to maintain cell line (e.g., use DMEM, if the cell line is maintained in DMEM medium).
- 50% FBS
- 40% complete growth medium
- 10% DMSO
Note: All operations must be done under sterile condition. Wipe carefully surface of reagent containing bottles (e.g., DMSO bottle, FBS bottle) with 70% ethanol before placing them inside flow hood.
Tips: You can also use sterile containers like beaker. We recommend you to use 50 ml sterile polypropylene tube.
Precautions: Do not store DMSO on ice. It will solidify on ice.
Tips: Freezing and thawing multiple times can cause proteins denaturation and precipitation.
- Serum-containing freezing medium is used for cryopreservation of cell lines.
Follow the table to prepare freezing medium of different volume.
|Reagent/volume||10 ml||50 ml||100 ml||250 ml|