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.

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.