3 Methods
3.1 Cooling
3.1.1 Passive Coolers
1. Prior to cryopreservation the passive cooler should be stored in
a 4
C refrigerator. To prepare, fill the system with coolant
(usually propanol) if required and place the device in a 4
C
fridge for at least 1 h before use, to allow for temperature
equilibration.
2. Prepare biological samples in the chosen cryoprotectant
medium and load them into a suitable cryocontainer. Transfer
promptly to the passive cooler to minimize any toxicity (see
Notes 4 and 5). Fill unused spaces in the cooler with vials
containing cryoprotectant medium only (see Note 6) to ensure
more consistent cooling within the device.
3. Place the passive cooler into a À80
C mechanical freezer (see
Notes 6–8). It is recommended that the cooler is placed
toward the back or bottom of any freezer, where thermal
variations are minimal, and that a chest (top opening) freezer
is used, as opening the door will cause less thermal variation
here than in a side-opening freezer.
4. During the cooling process, it is important that the mechanical
freezer remains closed as even brief opening will disrupt the
cooling cycle.
5. When the cooling run is complete, transfer the biological
samples to long-term storage below À120
C. The samples
should be thermally protected during this transfer as they can
warm rapidly, risking a negative impact on post-thaw outcome
(see Note 3). For a transport time of only a few minutes, leaving
the cryocontainers in the passive cooler will protect them from
excessive warming. For longer times, or delicate samples, transfer on dry ice or insulated chilled container for transport is
required (see Note 3).
3.1.2 Controlled-Rate
Freezers
1. Preparation of the freezer. As with passive coolers, it is recommended to prepare the freezing device prior to preparing the
cells. The chamber/sample holder of the CRF should be
pre-cooled to desired start temperature (see Note 9).
2. Selection of starting temperature. The starting temperature of
the cryopreservation protocol should be as low as possible to
reduce cryoprotectant toxicity and limit the overall cooling
time. It must, however, be above the freezing point of the
cryoprotective medium to avoid the risk of ice formation in
the sample before the commencement of the intended cooling
protocol, which could have damaging effects. Starting temperatures of +4
C to À2
C are typically used.
194
Peter Kilbride and Julie Meneghel
3.1 Cooling
3.1.1 Passive Coolers
1. Prior to cryopreservation the passive cooler should be stored in
a 4
C refrigerator. To prepare, fill the system with coolant
(usually propanol) if required and place the device in a 4
C
fridge for at least 1 h before use, to allow for temperature
equilibration.
2. Prepare biological samples in the chosen cryoprotectant
medium and load them into a suitable cryocontainer. Transfer
promptly to the passive cooler to minimize any toxicity (see
Notes 4 and 5). Fill unused spaces in the cooler with vials
containing cryoprotectant medium only (see Note 6) to ensure
more consistent cooling within the device.
3. Place the passive cooler into a À80
C mechanical freezer (see
Notes 6–8). It is recommended that the cooler is placed
toward the back or bottom of any freezer, where thermal
variations are minimal, and that a chest (top opening) freezer
is used, as opening the door will cause less thermal variation
here than in a side-opening freezer.
4. During the cooling process, it is important that the mechanical
freezer remains closed as even brief opening will disrupt the
cooling cycle.
5. When the cooling run is complete, transfer the biological
samples to long-term storage below À120
C. The samples
should be thermally protected during this transfer as they can
warm rapidly, risking a negative impact on post-thaw outcome
(see Note 3). For a transport time of only a few minutes, leaving
the cryocontainers in the passive cooler will protect them from
excessive warming. For longer times, or delicate samples, transfer on dry ice or insulated chilled container for transport is
required (see Note 3).
3.1.2 Controlled-Rate
Freezers
1. Preparation of the freezer. As with passive coolers, it is recommended to prepare the freezing device prior to preparing the
cells. The chamber/sample holder of the CRF should be
pre-cooled to desired start temperature (see Note 9).
2. Selection of starting temperature. The starting temperature of
the cryopreservation protocol should be as low as possible to
reduce cryoprotectant toxicity and limit the overall cooling
time. It must, however, be above the freezing point of the
cryoprotective medium to avoid the risk of ice formation in
the sample before the commencement of the intended cooling
protocol, which could have damaging effects. Starting temperatures of +4
C to À2
C are typically used.
194
Peter Kilbride and Julie Meneghel