have different water contents and CPA concentrations. To ensure
maximum permeation and homogeneous distribution of protective
molecules while minimizing the exposure time and toxicity effects,
it is needed to obtain insights in diffusion kinetics of water and
solutes. Various methods have been applied to study CPA permeation in tissues, including nuclear magnetic resonance [80, 81],
X-ray computer tomography [82, 83], and osmometer measurements [84, 85]. FTIR and Raman spectroscopy can be used to
study permeation and removal of individual CPA components in
mixtures as well as water fluxes [86–88].
6 Cryopreservation
6.1 Types
of Cryoprotective
Agent and CPA
Toxicity
A landmark paper by Polge and co-workers [89] reported that
addition of glycerol enables cock spermatozoa to survive freezing
and prolonged storage at À80
C. Glycerol is still widely used for
cryopreservation of sperm from different species [90] as well as
human red blood cells [91, 92]. Glycerol, however, appeared not to
be a universal cryoprotectant, and many cell types are difficult to
cryopreserve with glycerol. Lovelock and Bishop [93] reported the
cryoprotective properties of DMSO. Nowadays, DMSO is the most
widely used CPA for many types of mammalian as well as plant cells.
Examples of other types of permeating small molecular size CPAs
that have been identified include ethylene glycol, propylene glycol,
and dimethyl formamide [42]. A complete cryopreservation formulation typically consists of physiological salts, buffering components, nutrients, permeating CPAs, non-permeating agents,
antioxidants, and scavengers.
Why for some cell types one protectant works better than
others remains unclear. One factor that is thought to play an
important role is the cell membrane permeability to water and
CPAs, and their relative difference (see Fig. 4b). The CPA concentration also has to be considered, since CPA toxicity typically
increases with increasing concentration (see Fig. 5a).
CPA-membrane interactions can sometimes destabilize cellular
membranes, determining the suitability or unsuitability of a CPA
for a given cell type. DMSO interacts fundamentally different with
membranes compared to glycerol; DMSO increases the membrane
phase transition temperature, whereas glycerol decreases the membrane phase transition temperature [94, 95].
6.2 Slow Cooling
Cryopreservation
and Optimal
Cooling Rate
Cryopreservation requires CPAs; however also the cooling and
warming rate as well as the ice nucleation temperature determine
cryopreservation outcome. The cooling rate controls the extent
and rate of cellular dehydration during freezing. Moreover, the
rate of water transport across the cell membrane is dependent on
the temperature, and hence, indirectly, this affects the probability of
Principles of Cryopreservation and Freeze-Drying
17
maximum permeation and homogeneous distribution of protective
molecules while minimizing the exposure time and toxicity effects,
it is needed to obtain insights in diffusion kinetics of water and
solutes. Various methods have been applied to study CPA permeation in tissues, including nuclear magnetic resonance [80, 81],
X-ray computer tomography [82, 83], and osmometer measurements [84, 85]. FTIR and Raman spectroscopy can be used to
study permeation and removal of individual CPA components in
mixtures as well as water fluxes [86–88].
6 Cryopreservation
6.1 Types
of Cryoprotective
Agent and CPA
Toxicity
A landmark paper by Polge and co-workers [89] reported that
addition of glycerol enables cock spermatozoa to survive freezing
and prolonged storage at À80
C. Glycerol is still widely used for
cryopreservation of sperm from different species [90] as well as
human red blood cells [91, 92]. Glycerol, however, appeared not to
be a universal cryoprotectant, and many cell types are difficult to
cryopreserve with glycerol. Lovelock and Bishop [93] reported the
cryoprotective properties of DMSO. Nowadays, DMSO is the most
widely used CPA for many types of mammalian as well as plant cells.
Examples of other types of permeating small molecular size CPAs
that have been identified include ethylene glycol, propylene glycol,
and dimethyl formamide [42]. A complete cryopreservation formulation typically consists of physiological salts, buffering components, nutrients, permeating CPAs, non-permeating agents,
antioxidants, and scavengers.
Why for some cell types one protectant works better than
others remains unclear. One factor that is thought to play an
important role is the cell membrane permeability to water and
CPAs, and their relative difference (see Fig. 4b). The CPA concentration also has to be considered, since CPA toxicity typically
increases with increasing concentration (see Fig. 5a).
CPA-membrane interactions can sometimes destabilize cellular
membranes, determining the suitability or unsuitability of a CPA
for a given cell type. DMSO interacts fundamentally different with
membranes compared to glycerol; DMSO increases the membrane
phase transition temperature, whereas glycerol decreases the membrane phase transition temperature [94, 95].
6.2 Slow Cooling
Cryopreservation
and Optimal
Cooling Rate
Cryopreservation requires CPAs; however also the cooling and
warming rate as well as the ice nucleation temperature determine
cryopreservation outcome. The cooling rate controls the extent
and rate of cellular dehydration during freezing. Moreover, the
rate of water transport across the cell membrane is dependent on
the temperature, and hence, indirectly, this affects the probability of
Principles of Cryopreservation and Freeze-Drying
17