Preparation of freezing medium containing DMSO and FBS


  • 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.


  • Reagents
    • Fetal bovine serum (FBS) or Fetal calf serum (FCS)
    • DMSO (sterile)
    • Complete growth medium (optional)
  • Equipment and disposables
    • 50 ml sterile polypropylene tubes
    • Pipetboy
    • Pipets
    • 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


Preparation of 50 ml serum-containing freezing medium

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.


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.

Tips: You can also use sterile containers like beaker. We recommend you to use 50 ml sterile polypropylene tube.

Step 2: 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).
Note: Mixing of DMSO to serum is an exothermic reaction which can cause denaturation of serum proteins, resulting in precipitation. Therefore, it is recommended to use chilled FBS.

Precautions: Do not store DMSO on ice. It will solidify on ice.

Step 3 (optional): Check the sterility of freezing by keeping a small aliquot in a petri dish in the CO2 incubator.
Tips: We recommend you to check sterility of freezing medium.


Store at 4°C for few days. Freezing medium is stable for at least 6 months at -20°C.

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
FBS 5 25 50 125
DMSO 1 5 10 25
Growth medium 4 20 40 100