3. Selection of cooling rate. The importance of cooling rates in
cryopreservation is well-known and should be optimized for
each cell type (see Note 10) [5, 6, 13, 19]. These cooling rates
may be linear—i.e., one cooling rate throughout the cooling
process, or multistep, i.e., different cooling rates at different
steps in the protocol.
4. Loading of biological samples. Prepare biological samples in
chosen cryoprotectant medium at an appropriate temperature
(e.g., on ice) and load into suitable cryocontainers (see Note 4).
Transfer quickly to the CRF to minimize any toxicity (see Notes
4, 5, and 11). Start the cooling procedure.
5. Ice nucleation if required (see Subheading 3.2).
6. End temperature. An appropriate temperature must be chosen
where controlled cooling is no longer required, and samples
can be transferred to long-term storage (see Notes 3, 12, and
13). CRFs should stop cooling and maintain this final temperature until all biological samples have been removed. Programming a temperature a few degrees below the minimum
endpoint temperature will help protect the biologics from any
unintended warming during transfer to long-term storage (see
Notes 12 and 13).
7. When the cooling run is complete, transfer the biological
samples to long-term storage below À120
C. The samples
should be protected from the environment during this transfer,
as they can warm rapidly which will negatively impact postthaw outcomes. Transfer on dry ice or similar coolant may be
required (see Note 3).
3.2 Ice Nucleation
During Cooling
Manual nucleation can be used to nucleate a biological sample at a
defined temperature, which is beneficial for some cell types [20]. A
small section of the cryocontainer surface is cooled to significantly
below the equilibrium freezing point causing ice nucleation in the
limited, excessively supercooled region. Ice can then propagate
through the remainder of the sample at a relatively high temperature (see Note 14). Manual nucleation can be achieved using a
dedicated device such as a cryopen (a small device which, by expelling liquid nitrous oxide, can cool a small area to below À50
C
[21], see Notes 1 and 2) or the tips of pre-cooled forceps. Alternatively, an appropriate ice-nucleating agent can be included in the
cryocontainer (see Note 15). For manual nucleation:
1. Ensure that cryopreservation protocol includes a constant
temperature holding step approximately 5
C below the equilibrium freezing point of the cryopreservation medium
(see Note 6).
2. Maintain the holding temperature for at least 5 min to allow
biological sample temperature to equilibrate.
Control of Freezing Parameters
195
cryopreservation is well-known and should be optimized for
each cell type (see Note 10) [5, 6, 13, 19]. These cooling rates
may be linear—i.e., one cooling rate throughout the cooling
process, or multistep, i.e., different cooling rates at different
steps in the protocol.
4. Loading of biological samples. Prepare biological samples in
chosen cryoprotectant medium at an appropriate temperature
(e.g., on ice) and load into suitable cryocontainers (see Note 4).
Transfer quickly to the CRF to minimize any toxicity (see Notes
4, 5, and 11). Start the cooling procedure.
5. Ice nucleation if required (see Subheading 3.2).
6. End temperature. An appropriate temperature must be chosen
where controlled cooling is no longer required, and samples
can be transferred to long-term storage (see Notes 3, 12, and
13). CRFs should stop cooling and maintain this final temperature until all biological samples have been removed. Programming a temperature a few degrees below the minimum
endpoint temperature will help protect the biologics from any
unintended warming during transfer to long-term storage (see
Notes 12 and 13).
7. When the cooling run is complete, transfer the biological
samples to long-term storage below À120
C. The samples
should be protected from the environment during this transfer,
as they can warm rapidly which will negatively impact postthaw outcomes. Transfer on dry ice or similar coolant may be
required (see Note 3).
3.2 Ice Nucleation
During Cooling
Manual nucleation can be used to nucleate a biological sample at a
defined temperature, which is beneficial for some cell types [20]. A
small section of the cryocontainer surface is cooled to significantly
below the equilibrium freezing point causing ice nucleation in the
limited, excessively supercooled region. Ice can then propagate
through the remainder of the sample at a relatively high temperature (see Note 14). Manual nucleation can be achieved using a
dedicated device such as a cryopen (a small device which, by expelling liquid nitrous oxide, can cool a small area to below À50
C
[21], see Notes 1 and 2) or the tips of pre-cooled forceps. Alternatively, an appropriate ice-nucleating agent can be included in the
cryocontainer (see Note 15). For manual nucleation:
1. Ensure that cryopreservation protocol includes a constant
temperature holding step approximately 5
C below the equilibrium freezing point of the cryopreservation medium
(see Note 6).
2. Maintain the holding temperature for at least 5 min to allow
biological sample temperature to equilibrate.
Control of Freezing Parameters
195