2 Materials
2.1 Cooling
1. Biological sample—For optimal cryopreservation of a cell suspension, it is important to optimize the cryoprotectant(s) and
cooling rate for the specific cellular system [13–15]. These have
been discussed in detail elsewhere, and we point the reader to
the works referenced and theme 4 of this text.
2. Cryocontainer—Cryovials (typically <5 mL volume) are constructed of plastic with a flexible neck ring to relieve pressures
during cooling and warming. They are widely used, particularly
in research, but for the storage of medical-grade therapies,
hermetically sealed cryocontainers are required [16]. These
fall into three broad categories—cryobags, sealed vials, and
straws [16, 17]. These latter containers are also common for
the cryopreservation of small (<0.3 mL) biological samples
such as sperm, eggs, and embryos [16]. It is important to use
cryocontainers designed with specific structural properties for
safe use at ultra-low temperatures, as inappropriate plastics, for
example, can become brittle and shatter on warming.
3. Freezing devices—The three main choices for cryopreservation
are a passive cooler, a controlled-rate freezer (CRF) using LN 2 ,
or a CRF that is LN 2 -free [1–4]. When small sample numbers
are involved and viability can be recovered from rapid postthaw cell growth, passive coolers can be a practical option. They
provide an opportunity for low-cost, low-footprint cryopreservation and can be particularly appropriate in a research environment where cryopreservation is an enabling technology.
4. Both LN 2 and LN 2 -free CRFs provide controlled, accurate,
and reproducible cooling. A choice between them may rest,
for example, on cooling rates required (LN 2 devices have
higher maximum rate), available infrastructure and resources,
and the work location (LN 2 freezers cannot be operated in
cleanrooms) [4, 16, 18].
2.2 Ice Nucleation
During Cooling
1. Samples cooled to just below their equilibrium freezing point.
2. Cryopen (see Note 1) or pre-chilled forceps (see Note 2).
2.3 Storage
and Transfer
1. Liquid nitrogen vapor-phase storage tank.
2. Ultra-cold mechanical freezer.
3. Dry ice (see Note 3) or insulated chilled container.
2.4 Thawing
1. Thawing system.
2. Cryopreserved samples.
3. Prepared culture medium or washing solution.
4. Dry ice (see Note 3) or insulated chilled container.
Control of Freezing Parameters
193
2.1 Cooling
1. Biological sample—For optimal cryopreservation of a cell suspension, it is important to optimize the cryoprotectant(s) and
cooling rate for the specific cellular system [13–15]. These have
been discussed in detail elsewhere, and we point the reader to
the works referenced and theme 4 of this text.
2. Cryocontainer—Cryovials (typically <5 mL volume) are constructed of plastic with a flexible neck ring to relieve pressures
during cooling and warming. They are widely used, particularly
in research, but for the storage of medical-grade therapies,
hermetically sealed cryocontainers are required [16]. These
fall into three broad categories—cryobags, sealed vials, and
straws [16, 17]. These latter containers are also common for
the cryopreservation of small (<0.3 mL) biological samples
such as sperm, eggs, and embryos [16]. It is important to use
cryocontainers designed with specific structural properties for
safe use at ultra-low temperatures, as inappropriate plastics, for
example, can become brittle and shatter on warming.
3. Freezing devices—The three main choices for cryopreservation
are a passive cooler, a controlled-rate freezer (CRF) using LN 2 ,
or a CRF that is LN 2 -free [1–4]. When small sample numbers
are involved and viability can be recovered from rapid postthaw cell growth, passive coolers can be a practical option. They
provide an opportunity for low-cost, low-footprint cryopreservation and can be particularly appropriate in a research environment where cryopreservation is an enabling technology.
4. Both LN 2 and LN 2 -free CRFs provide controlled, accurate,
and reproducible cooling. A choice between them may rest,
for example, on cooling rates required (LN 2 devices have
higher maximum rate), available infrastructure and resources,
and the work location (LN 2 freezers cannot be operated in
cleanrooms) [4, 16, 18].
2.2 Ice Nucleation
During Cooling
1. Samples cooled to just below their equilibrium freezing point.
2. Cryopen (see Note 1) or pre-chilled forceps (see Note 2).
2.3 Storage
and Transfer
1. Liquid nitrogen vapor-phase storage tank.
2. Ultra-cold mechanical freezer.
3. Dry ice (see Note 3) or insulated chilled container.
2.4 Thawing
1. Thawing system.
2. Cryopreserved samples.
3. Prepared culture medium or washing solution.
4. Dry ice (see Note 3) or insulated chilled container.
Control of Freezing Parameters
193