Technical Documents
Nunc Tech Support Index
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Cryopreservation of Mammalian Cells
– Protocols
Vivi Kielberg, Kielberg Consult ApS
Most mammalian cells can be stored at temperatures below –130°C for many
years*. The viability of the cells after cryopreservation depends on their ability to cope
with the variety of stresses imposed on them during the freezing and thawing procedures.
The protocols given in this TechNote will be suitable for most cells. Minor adjustments
are always cell line dependent and therefore in each case must be found emperically.*) Nunc CryoTube™ Vials are recommended by ECACC (European
Collection of Animal Cell Cultures) for shipping and depositing cell lines (cf ECACC
general catalogue and ECACC shipping instructions).
Protocol for freezing of cells
- Cells to be frozen must not be infected with microorganisms (bacteria, yeast, mold,
mycoplasma and in special cases virus). In addition the cells must be viable and in
exponential growth.
- Gently harvest the cells. Spin down the cells as gently as possible (should not exceed
400 x g). Resuspend the cells in growth medium at
room temperature to a concentration of 2x106–2x107
cells per ml. Count the viable cells. Cells grown in protein free (or serum free) medium
cannot be expected to survive cryopreservation in their growth medium. Protein (e.g. BSA
or serum) must be added to the medium. After thawing, the cells can then be returned to
the required protein free medium.
- To avoid damage to the cell during freezing, a cryoprotectant is added to the growth
medium in which the cells are to be frozen. Glycerol or DMSO (dimethyl sulphoxide) in 10%
concentrations is most commonly used. Glycerol is non-toxic to the cells (and to the
personnel) and can be added directly to the cells. DMSO enters the cells more rapidly than
glycerol. DMSO is toxic to the cells (and to the personnel) if high concentrations are
used and if the cells are exposed to DMSO for prolonged periods (e.g. 10% DMSO for several
hours at room temperature). Because heat is generated when DMSO is dissolved in aqueous
solutions, it cannot be added directly to the cells. DMSO must be diluted to e.g. 20% in
medium, allowed to cool to below 37°C and subsequently added to the cells to a final
concentration of 10%.
- Mix cells and cryoprotectant at room temperature to final concentration of viable cells
in the range between 10
6 and 107 cells per ml and final concentration of cryoprotectant of 10%.
Cells frozen in lower or higher cell concentration often tend to have less viability.
Allow the cryoprotectant to enter the cells. At least 20 minutes and, for DMSO, not more
than 30 minutes should pass before the cooling procedure is started. This is usually the
time it takes to aliquot the cells into CryoTube™ Vials.
If a uniform batch of cells where every aliquot is representative of the whole batch is
required, all the cells must be collected at this step into one vial and properly mixed,
before aliquoting is performed. Usually 1 ml aliquots are used, but other volumes can also
be used. As 1 ml cryo vials are very small, it is advisable to use sterile gloves when
opening and closing the vials in order to minimize the risk of contamination during
aliquoting. Care should be taken not to exceed the maximum filling volume of the CryoTube
vial. CryoTube vials made of polypropylene are most commonly used for storage of the
cells. When cells are to be stored in the vapour phase of liquid nitrogen or in a
–140°C mechanical freezer, CryoTube vials with internal thread and silicone gasket
are the safest to use. This kind of cryo vial has the best seal and, provided that the
vial is properly closed (neither too loose nor too tight), there is no risk of
contamination with microorganisms during storage. When cells are to be
stored in the liquid phase of liquid nitrogen it is recommended to enclose
the CryoTube™ Vials in heat sealable Nunc CryoFlex™ tube wrap. Be careful
not to heat the vial excessively as this will injure the cells. If the cryo vials contain
hazardous material – no matter how they are stored – the vials must be sealed
with Nunc CryoFlex tube wrap to reduce the risk for the personnel handling the CryoTube
vials.
The most common cooling rate used is 1°C per minute in the range from room temperature
and down to below –50°C. Once the temperature is below –50°C, the CryoTube
vials can be submerged directly in liquid nitrogen (–196°C). Special equipment for
precise controlling of the cooling rates is available on the market. Such equipment is
very expensive and usually only needed for very sensitive cells.
In most cases the cooling rate can be controlled in a less expensive manner:
As a rule of thumb, a –70°C mechanical freezer cools a litre cube of water
1°C/minute.
To achieve a similar cooling rate, the cryo vials can be placed in an insulated cube of
approximately 1 litre capacity. The insulation can be paper, cotton, wool, or styrofoam.
In theory, cells can be stored indefinitely below
–130°C, as no biological process takes place below this temperature. In practice,
background ionizing radiation will in time (decades) injure the cells. Cells stored for
prolonged periods above –130°C tend to be less stable.
Be aware that cells stored in small liquid nitrogen tanks without automatic refill may not
be stored permanently below –130°C if the tanks are opened frequently. The
temperature at the topof the tank rises as the nitrogen evaporates from the tank.
Therefore, a temperature gradient is established from the surface of the liquid nitrogen
towards the top of the tank.
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Protocol for thawing of cells
- To obtain the best possible survival of the cells, thawing
of the cells must be performed as quickly as possible. Once the CryoTube vial is removed
from the freezer/liquid nitrogen tank, it should be placed directly into a 37°C water
bath and shaken until it is completely thawed. If liquid nitrogen has entered the CryoTube
vial, it will explode upon warming, because liquid nitrogen will expand 700 times during
the warming process. Personnel should always wear protective clothes, glasses and gloves,
while handling CryoTube vials from liquid nitrogen tanks, and should transport the vial in
a covered, insulated bucket or box. An explosion may happen seconds after the vial has
been removed from the liquid nitrogen.
- To avoid transfer of microorganisms from the freezer/liquid
nitrogen tank or the water bath to the laminar flow cabinet and subsequently to the
culture, the CryoTube vials are soaked in 70% ethanol before they are transferred to the
laminar flow cabinet.
- If the cryo protectant used is glycerol, the cells can be
diluted 10 times directly into a TC flask or TC dish. If DMSO is used, the cells should be
washed once in growth medium before being added to the TC flask in fresh growth medium.
Some cells are very sensitive at this stage of the procedure, and it may be advisable to
dilute the DMSO stepwise to minimize the osmotic stress imposed upon the cells when DMSO
is diluted. It is advisable to take out a sample of the thawed cells for a viability test
before the cells are placed in the incubator.
- In order to avoid physical damage of cells that form a
monolayer, such cultures should be left untouched in the incubator for at least 16 hours
before assessing the result.
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Vivi Kielberg, Kielberg Consult ApS,
Frændevej 16, DK-2860 Søborg, Denmark |
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