important as the earlier cooling process. Immersion of cryocontainers in a warmed water bath is the established, and probably still
most common, method of thawing cryopreserved biological samples. In the past decade, dry thawing systems have become more
common due to lower contamination risks and greater consistency.
3.4.1 Water Bath
1. Warm water bath to 37
C (see Note 18).
2. Remove the biological sample from storage device, being careful not to warm other samples in the same storage location.
3. Place sample in water bath, and where possible avoid wetting
the lid or inlet/outlet ports of the cryocontainer. Use of a
submerged rack or floating vial holder is often useful here.
4. Visually inspect the sample, and when only a small ice crystal
remains visible, remove the sample and quickly begin any
washing or post-thaw steps [23]. As a rule of the thumb,
2 mL cryovials containing 1 mL of liquid take ~ 2 min to
thaw from approximately À120
C in a 37
C water bath (see
Note 19).
3.4.2 Dry Thawing
Systems
1. Ensure that the correct thawing system is used—some are
optimized for vials, others cryobags, and some both.
2. Remove the biological sample from storage, being careful not
to warm other samples within the same storage location.
3. Place sample in the equipment for the required time.
4. Visually inspect biological sample when thawing is believed to
be complete, to ensure no more than a small ice crystal remains.
5. Promptly start any washing or post-thaw steps [23].
4 Notes
1. A cryopen is a small device (in shape and size like a pen),
containing nitrous oxide under pressure. When a small volume
of the gas is released onto the outer surface of the sample
container, a transient and localized region of deep cooling is
generated that cools the inner container surface sufficiently to
initiate ice nucleation.
2. In the absence of a cryopen, it is possible to use some
pre-chilled forceps or other metal surface to nucleate ice.
Pre-chilling can be done by dipping the tip of the forceps in
LN 2 and then used in the same way as a cryopen by touching
the side of the cryocontainer with them. The forceps or metal
implement should be re-cooled between samples, as these will
warm rapidly in the air.
Control of Freezing Parameters
197
most common, method of thawing cryopreserved biological samples. In the past decade, dry thawing systems have become more
common due to lower contamination risks and greater consistency.
3.4.1 Water Bath
1. Warm water bath to 37
C (see Note 18).
2. Remove the biological sample from storage device, being careful not to warm other samples in the same storage location.
3. Place sample in water bath, and where possible avoid wetting
the lid or inlet/outlet ports of the cryocontainer. Use of a
submerged rack or floating vial holder is often useful here.
4. Visually inspect the sample, and when only a small ice crystal
remains visible, remove the sample and quickly begin any
washing or post-thaw steps [23]. As a rule of the thumb,
2 mL cryovials containing 1 mL of liquid take ~ 2 min to
thaw from approximately À120
C in a 37
C water bath (see
Note 19).
3.4.2 Dry Thawing
Systems
1. Ensure that the correct thawing system is used—some are
optimized for vials, others cryobags, and some both.
2. Remove the biological sample from storage, being careful not
to warm other samples within the same storage location.
3. Place sample in the equipment for the required time.
4. Visually inspect biological sample when thawing is believed to
be complete, to ensure no more than a small ice crystal remains.
5. Promptly start any washing or post-thaw steps [23].
4 Notes
1. A cryopen is a small device (in shape and size like a pen),
containing nitrous oxide under pressure. When a small volume
of the gas is released onto the outer surface of the sample
container, a transient and localized region of deep cooling is
generated that cools the inner container surface sufficiently to
initiate ice nucleation.
2. In the absence of a cryopen, it is possible to use some
pre-chilled forceps or other metal surface to nucleate ice.
Pre-chilling can be done by dipping the tip of the forceps in
LN 2 and then used in the same way as a cryopen by touching
the side of the cryocontainer with them. The forceps or metal
implement should be re-cooled between samples, as these will
warm rapidly in the air.
Control of Freezing Parameters
197