to occur when single components are used at high concentrations.
The cooling rate has to be fast enough to avoid ice formation;
however there is a tradeoff between cooling rate and CPA toxicity
(see Fig. 5a). If high CPA concentrations are used, slower cooling
rates can be used to avoid ice formation. On the other hand, lower
concentrations are less toxic, but require higher cooling rates, and
hence can only be used for small samples.
Achieving rapid enough warming rates is one of the main
challenges for both cryopreservation and vitrification approaches,
particularly when low amounts of CPAs are used to reduce toxicity,
and for larger specimens such as organs. Various approaches have
been developed that can be used for (ultra-)rapid and homogeneous warming of cryopreserved samples including laser warming
[100], nanowarming [101], and electromagnetic heating [102].
7 Lyopreservation by Freeze-Drying
7.1 Freeze-Drying
Freeze-drying is the prefered method for drying of biological samples, because it is a relatively robust and reproducible procedure.
During freeze-drying first the sample is frozen to immobilize all
solution components. This can be done on the shelves of the freezedryer, or in an external (controlled rate) freezer. During primary
drying, ice is removed from the sample via sublimation. This is done
by lowering the vacuum pressure in the drying chamber to a value
below that of the vapor pressure of ice (i.e., at the sample temperature), such that water will migrate from the sample to the condenser. During primary drying, the sample temperature must be
maintained below the eutectic, glass transition, collapse, and/or
melt temperature, to minimize sample damage during drying
[103]. After removal of bulk water, a secondary drying stage is
used to remove residual moisture still adsorbed to the sample by
desorption, which is done by supplying heat to the sample. Thereafter, samples can be returned to ambient conditions. To increase
longevity, storage can be done under vacuum or under reduced
oxygen and/or low relative humidity conditions.
7.2 Formulations
for Freeze-Drying
of Cells
Freeze-drying of cells requires protective agents to protect during
both freezing and drying. Freeze-drying formulations for cells
comprise of a suitable physiological buffer solution, a protective
agent like sucrose or trehalose, and a bulking agent. Proteins are
typically added as bulking agent to space the cells and to increase
the T g of the formulation. As indicated above, special measures can
be employed to introduce protective disaccharides into the cells,
but also freezing itself results in uptake of disaccharides. Albumin in
combination with trehalose, added at, respectively, 5% (w/v) and
150–300 mM to the cell solution, has proven to be a stable matrix
for cells [74, 104]. DNA in freeze-dried sperm protected with
Principles of Cryopreservation and Freeze-Drying
19
The cooling rate has to be fast enough to avoid ice formation;
however there is a tradeoff between cooling rate and CPA toxicity
(see Fig. 5a). If high CPA concentrations are used, slower cooling
rates can be used to avoid ice formation. On the other hand, lower
concentrations are less toxic, but require higher cooling rates, and
hence can only be used for small samples.
Achieving rapid enough warming rates is one of the main
challenges for both cryopreservation and vitrification approaches,
particularly when low amounts of CPAs are used to reduce toxicity,
and for larger specimens such as organs. Various approaches have
been developed that can be used for (ultra-)rapid and homogeneous warming of cryopreserved samples including laser warming
[100], nanowarming [101], and electromagnetic heating [102].
7 Lyopreservation by Freeze-Drying
7.1 Freeze-Drying
Freeze-drying is the prefered method for drying of biological samples, because it is a relatively robust and reproducible procedure.
During freeze-drying first the sample is frozen to immobilize all
solution components. This can be done on the shelves of the freezedryer, or in an external (controlled rate) freezer. During primary
drying, ice is removed from the sample via sublimation. This is done
by lowering the vacuum pressure in the drying chamber to a value
below that of the vapor pressure of ice (i.e., at the sample temperature), such that water will migrate from the sample to the condenser. During primary drying, the sample temperature must be
maintained below the eutectic, glass transition, collapse, and/or
melt temperature, to minimize sample damage during drying
[103]. After removal of bulk water, a secondary drying stage is
used to remove residual moisture still adsorbed to the sample by
desorption, which is done by supplying heat to the sample. Thereafter, samples can be returned to ambient conditions. To increase
longevity, storage can be done under vacuum or under reduced
oxygen and/or low relative humidity conditions.
7.2 Formulations
for Freeze-Drying
of Cells
Freeze-drying of cells requires protective agents to protect during
both freezing and drying. Freeze-drying formulations for cells
comprise of a suitable physiological buffer solution, a protective
agent like sucrose or trehalose, and a bulking agent. Proteins are
typically added as bulking agent to space the cells and to increase
the T g of the formulation. As indicated above, special measures can
be employed to introduce protective disaccharides into the cells,
but also freezing itself results in uptake of disaccharides. Albumin in
combination with trehalose, added at, respectively, 5% (w/v) and
150–300 mM to the cell solution, has proven to be a stable matrix
for cells [74, 104]. DNA in freeze-dried sperm protected with
Principles of Cryopreservation and Freeze-Drying
19