3. Any suitably cooled and insulated container is effective for
transfer. If no dry ice is available, cold-box gel kits pre-cooled
to À80
C or below can be used, as well as any specific coldtransfer device which can maintain the endpoint temperature or
lower. Using a small Dewar containing liquid nitrogen is not
advisable due to the possibility of LN 2 ingress and the risks of
spills and splashes of LN 2 from carrying open containers (see
Note 17).
4. When transferring procedure-ready samples on ice, it is important to ensure that the cryocontainer is dried immediately
before loading into the cooling device. Any water remaining
on the outside of the sample will freeze on cooling and may
result in the sample being stuck to the cooler when it reaches its
target temperature.
5. When labelling cryocontainers, it is critically important to use
ink and materials which are resistant to both low temperatures
and water. Labels designed for only ambient temperatures can
become brittle and lose their adhesive properties during ultralow temperature storage.
6. In this chapter we have at several points recommended that a
“dummy” vial or cryocontainer, containing cryoprotectant
medium but no cells, is used. This can be set up to record
the actual temperature within the cryocontainer that, due to
the thermal mass of the sample and the physical properties of
the cryocontainer, will lag behind the device programmed
temperature. Once the actual sample temperatures have been
established, the programmed cryopreservation protocol can be
modified as required. The process can be carried out without
using dummy cryocontainers in every run as the cooling/thawing process should be consistent when carefully repeated, but
for maximum security, a dummy cryocontainer could be
included in each run. For dummy cryocontainer thermocouple
recommendations (see Note 8).
7. To alter cooling rates in a passive cooler, a range of mechanical
freezers can be used. For example, using a À150
C freezer will
increase the cooling rate by approximately 70% compared to
À80
C system. When changing cooling environment, using a
thermocouple in a vial containing cryoprotectant medium to
test actual cooling rates is useful (see Notes 6 and 8). While
many passive coolers claim a cooling rate of 1
C/min when
placed into a À80
C environment, this is a calculated, mean
value and will only be achieved for a small part of the cooling
process. It will be impacted by sample load and freezer temperature fluctuations.
8. When setting up a dummy cryocontainer, it is recommended
that a thermocouple attached to a data logger is used. The
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Peter Kilbride and Julie Meneghel
transfer. If no dry ice is available, cold-box gel kits pre-cooled
to À80
C or below can be used, as well as any specific coldtransfer device which can maintain the endpoint temperature or
lower. Using a small Dewar containing liquid nitrogen is not
advisable due to the possibility of LN 2 ingress and the risks of
spills and splashes of LN 2 from carrying open containers (see
Note 17).
4. When transferring procedure-ready samples on ice, it is important to ensure that the cryocontainer is dried immediately
before loading into the cooling device. Any water remaining
on the outside of the sample will freeze on cooling and may
result in the sample being stuck to the cooler when it reaches its
target temperature.
5. When labelling cryocontainers, it is critically important to use
ink and materials which are resistant to both low temperatures
and water. Labels designed for only ambient temperatures can
become brittle and lose their adhesive properties during ultralow temperature storage.
6. In this chapter we have at several points recommended that a
“dummy” vial or cryocontainer, containing cryoprotectant
medium but no cells, is used. This can be set up to record
the actual temperature within the cryocontainer that, due to
the thermal mass of the sample and the physical properties of
the cryocontainer, will lag behind the device programmed
temperature. Once the actual sample temperatures have been
established, the programmed cryopreservation protocol can be
modified as required. The process can be carried out without
using dummy cryocontainers in every run as the cooling/thawing process should be consistent when carefully repeated, but
for maximum security, a dummy cryocontainer could be
included in each run. For dummy cryocontainer thermocouple
recommendations (see Note 8).
7. To alter cooling rates in a passive cooler, a range of mechanical
freezers can be used. For example, using a À150
C freezer will
increase the cooling rate by approximately 70% compared to
À80
C system. When changing cooling environment, using a
thermocouple in a vial containing cryoprotectant medium to
test actual cooling rates is useful (see Notes 6 and 8). While
many passive coolers claim a cooling rate of 1
C/min when
placed into a À80
C environment, this is a calculated, mean
value and will only be achieved for a small part of the cooling
process. It will be impacted by sample load and freezer temperature fluctuations.
8. When setting up a dummy cryocontainer, it is recommended
that a thermocouple attached to a data logger is used. The
198
Peter Kilbride and Julie Meneghel