Technical Documents
Nunc Tech Support Index
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Aspects of Cryo Preservation
A variety of types and brands of vials are used for the storage of biological materials at
low temperature.
The purpose of storage at low temperature is to keep and maintain materials
for later use, e.g. to preserve original cells for later culture.
It is of great importance that the storage vials do not cause any change or
damage to the sample, which is often irreplaceable.
When choosing a suitable vial or container for cryo preservation, the
following aspects should be considered: |
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- Tightness
- Toxicity
- Mutagenicity
- Pyrogenicity
- Sterility
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Fig.5:
CryoFlex™ protective tubing used as a "cane"
for lowering CryoTubes™ into liquid nitrogen. |
| Tightness |
For long term storage of samples, particularly in liquid
nitrogen, a completely leak proof seal is needed.
If liquid nitrogen enters the vial during storage, the vial may explode when
it is thawed due to vaporization of the liquid nitrogen inside the vial. When heated to
room temperature, liquid nitrogen will expand about 700 times, or create a pressure of
about 700 atmospheres if expansion is prohibited!
At ultralow temperatures, any sealing material becomes inflexible, so there is
always a theoretical risk of leakage when a mechanical closure is used.
To avoid the risk of explosion of vials which have been stored in liquid
nitrogen, they may be kept in the gas phase in the nitrogen container for a minimum of 24
hours before removal to higher temperatures. This will allow any liquid nitrogen present
to boil off slowly thus preventing the dramatic expansion as described above.
To ensure 100% leak proof storage, mechanically closed containers must be
hermetically sealed in an outer protective envelope.
The Nunc CryoSystem includes heat-sealable polyethylene tubing, CryoFlex
(Cat.No. 343958), for hermetic enveloping of CryoTubes (Fig. 5).
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| Toxicity |
In order to preserve the viability of living samples, such
as cultured cells, possibly for years, it is necessary that the vial material is
absolutely non-toxic.
Plastic vials are commonly used for storing such samples at low or ultralow
temperatures. However, the choice of plastic grade for these vials is very critical as
some grades may contain impurities or additives, which are toxic to cells.
When choosing vials for cryo preservation, proven non-toxicity of the vial
material is therefore of great importance.
Nunc CryoTubes have been proven non-toxic to animal cells by passing the
following tests:
- Growth inhibition test. (Ref.: Materials Science Toxicology
Laboratories, University of Tennessee, USA).
- Cloning inhibition test. (Ref.: Nunc Laboratories, 1985).
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| Mutagenicity |
As one purpose of storing samples, including cultured
cells, is to maintain a reference stock of unaltered material, consideration should be
given to the possible mutagenic effect of components which may be absorbed from the vial.
It should be noted that the effect of mutagenic components absorbed during
storage may only be expressed after a number of post-storage cell divisions, as
mutagenesis is a long term effect on life processes, which are brought to a standstill
during storage. Thus, the results of mutagenicity may not appear until the cells have been
subcultured several times.
Nunc CryoTubes have been proven non-mutagenic by passing the AmesTest. (Reg.:
OECD Salmonella typhimurium, Reverse Mutation Assay No. 471, 1983).
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| Pyrogenicity |
Cryo preservation vials may be used for the storage of
substances used in pharmacological studies.
Pyrogenic impurities released from the vial are unacceptable because they
could possibly interfere with the effects of pharmacological substances.
Endotoxins are known to be especially adsorptive to polypropylene, the type of
plastic used for cryo preservation vials. One should therefore pay special attention to
the possible presence of these pyrogens.
Nunc CryoTubes have been proven virtually free of endotoxins by passing the
Limulus Amebocyte Lysate (LAL) test with a value lower than required for water used for
injection, i.e. 0.25 Endotoxin Units/ml. (Ref.: USP XXI, 1985).
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| Sterility |
To maintain the integrity of the stored material, it must
be free from contamination with foreign micro-organisms.
A proven Sterility Assurance Level (SAL) is another important criterion for
the choice of vial.
Nunc CryoTubes have been subject to bioburden monitoring and subsequent
radiation sterilization to ensure a SAL of 10-6, i.e. statistically not more than one
surviving micro-organism per one million vials.
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Arne Johansson
Nunc Laboratories |
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