- The freezing medium is used to preserve cell lines at ultra-low temperatures. This method of preserving cell lines is called cryopreservation.
- Freezing medium is nothing but a growth medium supplemented with a high concentration of serum and a cryoprotective agent such as DMSO or glycerol.
- As the name suggests, cryoprotective agents protect cells from death at a temperature below the freezing point.
- Serum concentration as high as 90% can be used in the freezing medium. High serum concentration improves cell viability and recovery after thawing.
- Cryoprotective agents protect cells from death by preventing ice crystal formation.
- Serum-free chemically-defined freezing media are also available. They are very useful to preserve cell lines which are maintained in a serum-free chemically defined medium.
- Both serum-containing and serum-free freezing medium are available commercially.
- A Serum-containing freezing medium is used for cell lines which are maintained in a serum-supplemented growth medium. We recommend checking the cell line manual for the optimal freezing medium.
> Serum *
> DMSO (sterile)
> Complete growth medium
* Choose the serum which is used for the preparation of growth medium. For example, use FBS if you have used FBS for the preparation of growth medium.
Equipment and disposables
> 50 ml sterile polypropylene tubes
> Laminar flow hood
> 50% FBS
> 10% DMSO
> 40% growth medium
Preparation of 50 ml serum-containing freezing medium
All operations must be done under sterile conditions. Wipe carefully the surfaces of all reagent-containing bottles (e.g., DMSO bottle, FBS bottle) with 70% ethanol before placing them inside the laminar flow hood.
Step 1: To prepare 50 ml freezing medium, transfer 25 ml FBS and 20 ml growth medium to a 50-ml sterile polypropylene tube. Tighten the cap of the tube and mix by gentle inversion. Store it on ice.
You can also use sterile containers like beakers. We recommend you to use a 50 ml sterile polypropylene tube.
Step 2: Add 5 ml DMSO slowly while shaking the tube. Tighten the cap of the tube and mix by inverting the tube many times (5 – 6 times).
Mixing of DMSO to serum is an exothermic reaction that can cause denaturation of serum proteins, resulting in precipitation. Therefore, it is recommended to use chilled FBS.
Do not store DMSO on ice. It will solidify on ice.
Step 3 (optional): Check the sterility of freezing media by keeping a small aliquot in a petri dish in the CO2 incubator.
We recommend you to check the sterility of the freezing medium.
Store at 4°C for a few days. The freezing medium is stable for at least 6 months at -20°C.
Freezing and thawing multiple times can cause protein denaturation and precipitation.
Serum-containing freezing medium is used for cryopreservation of cell lines.
|Follow the table to prepare a freezing medium of different volumes.|
|Reagent/volume||10 ml||50 ml||100 ml||250 ml|
|* Use appropriate serum e.g., use FBS if the growth medium contains FBS.|