Preparation of Freezing Medium Containing DMSO and Serum

Overview

  • 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.
Freezing Medium Types
Types of Freezing Media

Requirements

Reagents
> 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
> Pipetboy
> Pipette
> Laminar flow hood

Composition
> 50% FBS
>
10% DMSO
> 40% growth medium

Objective
Preparation of 50 ml serum-containing freezing medium

Note
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.

Preparation

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.

Tip:
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).

Note:
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.

Caution:
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.

Tip:
We recommend you to check the sterility of the freezing medium.

Storage:
Store at 4°C for a few days. The freezing medium is stable for at least 6 months at -20°C.

Caution:
Freezing and thawing multiple times can cause protein denaturation and precipitation.

Applications
Serum-containing freezing medium is used for cryopreservation of cell lines.

Follow the table to prepare a freezing medium of different volumes.
Reagent/volume10 ml50 ml100 ml250 ml
Serum *52550125
DMSO151025
Growth medium42040100
* Use appropriate serum e.g., use FBS if the growth medium contains FBS.

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