thermocouple lead should be small and flexible enough to be
inserted into a vial or other cryocontainer easily. The entry
point must be securely sealed. The thermocouple junction
(at the end of the lead) should be in the center of the liquid
of small vials (<2 mL), and several spatially separated thermocouples may be used in larger containers. Many types of thermocouple are available, and type T can be recommended due to
their better accuracy at low temperatures when compared to
other common types.
9. When loading and cooling cryocontainers such as vials, it is
important that they are upright for consistency. Cryobags
should be cryopreserved with as few air bubbles as possible,
as these insulate the biological sample on both cooling and
thawing, leading to suboptimal outcome.
10. For most somatic mammalian cells in suspension, cooling rates
of 1
C/min are usually a good starting point for optimization
studies [24]. For larger cell structures, such as spheroids and
organoids, the longer time required for dehydration suggests
that a slower rate such as 0.3
C/min is a better starting point.
Some specific cell types, such as sperm cells and erythrocytes,
require relatively fast cooling rates (10
C/min or greater).
11. When cooling multiple samples in one cooling run in an LN 2 -
based CRF, it is advisable to leave a gap between individual
cryocontainers. These freezers cool by blowing cold nitrogen
into the freezing chamber at a specific rate. This can result in
differing cooling rates between those samples directly exposed
to this cold nitrogen and those that may be partially insulated
by other samples surrounding it. For sensitive cells this can lead
to variable and/or suboptimal results on thaw.
12. This endpoint temperature should always be below À50
C,
and À80
C is a commonly adopted final temperature. The
À80
C achieved when a passive cooler is placed into mechanical freezer is a convenient end temperature for such devices.
13. As the temperature of a biological sample temperature typically
lags behind freezer temperature, albeit slightly, it is preferable
to wait a few minutes after the freezer reaches its endpoint
temperature before transfer of the sample to long-term
storage.
14. Ice nucleation is volume dependent, and larger samples
(>2 mL) usually nucleate with minimal supercooling, whereas
smaller volumes are more susceptible to excessive supercooling. Where possible, biological sample volumes should be as
large as possible when dealing with known, nucleationsensitive material.
15. Some freezing protocols include an ice-nucleating agent in the
cryoprotective medium. These are reagents which are included
Control of Freezing Parameters
199
inserted into a vial or other cryocontainer easily. The entry
point must be securely sealed. The thermocouple junction
(at the end of the lead) should be in the center of the liquid
of small vials (<2 mL), and several spatially separated thermocouples may be used in larger containers. Many types of thermocouple are available, and type T can be recommended due to
their better accuracy at low temperatures when compared to
other common types.
9. When loading and cooling cryocontainers such as vials, it is
important that they are upright for consistency. Cryobags
should be cryopreserved with as few air bubbles as possible,
as these insulate the biological sample on both cooling and
thawing, leading to suboptimal outcome.
10. For most somatic mammalian cells in suspension, cooling rates
of 1
C/min are usually a good starting point for optimization
studies [24]. For larger cell structures, such as spheroids and
organoids, the longer time required for dehydration suggests
that a slower rate such as 0.3
C/min is a better starting point.
Some specific cell types, such as sperm cells and erythrocytes,
require relatively fast cooling rates (10
C/min or greater).
11. When cooling multiple samples in one cooling run in an LN 2 -
based CRF, it is advisable to leave a gap between individual
cryocontainers. These freezers cool by blowing cold nitrogen
into the freezing chamber at a specific rate. This can result in
differing cooling rates between those samples directly exposed
to this cold nitrogen and those that may be partially insulated
by other samples surrounding it. For sensitive cells this can lead
to variable and/or suboptimal results on thaw.
12. This endpoint temperature should always be below À50
C,
and À80
C is a commonly adopted final temperature. The
À80
C achieved when a passive cooler is placed into mechanical freezer is a convenient end temperature for such devices.
13. As the temperature of a biological sample temperature typically
lags behind freezer temperature, albeit slightly, it is preferable
to wait a few minutes after the freezer reaches its endpoint
temperature before transfer of the sample to long-term
storage.
14. Ice nucleation is volume dependent, and larger samples
(>2 mL) usually nucleate with minimal supercooling, whereas
smaller volumes are more susceptible to excessive supercooling. Where possible, biological sample volumes should be as
large as possible when dealing with known, nucleationsensitive material.
15. Some freezing protocols include an ice-nucleating agent in the
cryoprotective medium. These are reagents which are included
Control of Freezing Parameters
199