3. Access the cryocontainers as rapidly as possible, and, holding
them so as not to further warm their content, use the cryopen
to cool the surface of the cryocontainer. This cooling should be
focused until ice is seen to nucleate. Be careful to cool only one
point of the vial (see Notes 2 and 16).
4. Ice nucleation will cause the cryocontainer contents to turn
from clear liquid to opaque ice.
5. Return the cryocontainer to the controlled-rate freezer as rapidly as possible and resume the cooling profile when all samples
have been nucleated.
6. If samples take longer than ~ 30 s to nucleate, return the
samples and reduce the device’s hold temperature a further
2
C.
3.3 Storage
and Transfer
Storage of biological samples containing live cells for any more than
a few days must be below the glass transition temperature of the
cryoprotectant medium [5, 8]. In DMSO-based cryoprotectants,
this is typically ~À120
C. Storage is therefore in LN 2 vapor or in an
ultra-low mechanical freezer. Appropriate safety considerations
should always be in place where this hardware is present. Inadequate handling during transfer between the cooling device and
long-term storage location can lead to damaging sample warming.
1. Ensure that the controlled rate or passive cooler has reached
the final temperature and that the biological samples have
equilibrated below at least À50
C [6, 22].
2. Prepare a polystyrene box containing dry ice or other appropriate cold-transfer system and ensure that space is available in the
long-term storage (see Note 3).
3. Remove cryocontainers by their lids, or area containing the
least biological material, and place quickly into the dry ice or
transfer vessel. Always use insulated gloves and other appropriate personal protective equipment.
4. Add biological samples to appropriate location in long-term
storage. It is essential to ensure that samples are positioned in
the storage container to minimize warming of surrounding,
already stored samples during retrieval of the required material
(see Note 17). Dispersal of duplicate samples to different locations in a storage tank is recommended, as is the use of duplicate tanks.
5. Maintain the freezer at the end temperature until transfer to
storage is complete. If there is a problem during transfer, then
samples can be temporarily returned to the cooling device.
3.4 Thawing
It is important to note that, for optimal results, correct thawing and
post-thaw treatment of a cryopreserved biological sample are as
196
Peter Kilbride and Julie Meneghel
them so as not to further warm their content, use the cryopen
to cool the surface of the cryocontainer. This cooling should be
focused until ice is seen to nucleate. Be careful to cool only one
point of the vial (see Notes 2 and 16).
4. Ice nucleation will cause the cryocontainer contents to turn
from clear liquid to opaque ice.
5. Return the cryocontainer to the controlled-rate freezer as rapidly as possible and resume the cooling profile when all samples
have been nucleated.
6. If samples take longer than ~ 30 s to nucleate, return the
samples and reduce the device’s hold temperature a further
2
C.
3.3 Storage
and Transfer
Storage of biological samples containing live cells for any more than
a few days must be below the glass transition temperature of the
cryoprotectant medium [5, 8]. In DMSO-based cryoprotectants,
this is typically ~À120
C. Storage is therefore in LN 2 vapor or in an
ultra-low mechanical freezer. Appropriate safety considerations
should always be in place where this hardware is present. Inadequate handling during transfer between the cooling device and
long-term storage location can lead to damaging sample warming.
1. Ensure that the controlled rate or passive cooler has reached
the final temperature and that the biological samples have
equilibrated below at least À50
C [6, 22].
2. Prepare a polystyrene box containing dry ice or other appropriate cold-transfer system and ensure that space is available in the
long-term storage (see Note 3).
3. Remove cryocontainers by their lids, or area containing the
least biological material, and place quickly into the dry ice or
transfer vessel. Always use insulated gloves and other appropriate personal protective equipment.
4. Add biological samples to appropriate location in long-term
storage. It is essential to ensure that samples are positioned in
the storage container to minimize warming of surrounding,
already stored samples during retrieval of the required material
(see Note 17). Dispersal of duplicate samples to different locations in a storage tank is recommended, as is the use of duplicate tanks.
5. Maintain the freezer at the end temperature until transfer to
storage is complete. If there is a problem during transfer, then
samples can be temporarily returned to the cooling device.
3.4 Thawing
It is important to note that, for optimal results, correct thawing and
post-thaw treatment of a cryopreserved biological sample are as
196
Peter Kilbride and Julie Meneghel