concentrations in the freeze-concentrated (non-frozen) solution
results in movement of intracellular water to the extracellular
phase, which causes cells to dehydrate. In addition to restoring
the osmotic equilibrium, this increases the concentration of intracellular molecules and constituents and brings organelles in close
proximity to each other. Solution effects injury refers to cell damage
due to cells being situated in a freeze-concentrated solution of
residual non-freezable water and salts [28, 29]. Cells can tolerate
freezing-induced dehydration to a considerable extent, but if certain tolerance limits are exceeded, viability is lost [26, 27].
Biomolecules (proteins, lipids, DNA, and RNA) may undergo
conformational and phase changes as a consequence of lowering
the temperature and freezing-induced dehydration. Especially
membrane phase changes and changes in the organization of membrane domains during cooling and freezing may cause malfunctioning after reconstitution [30, 31]. Macromolecular assemblies such
as liposomes have been described to suffer from fusion and leakage
during freezing [32].
3.2 Drying Injury
Freezing and drying are different stress factors [21]. In contrast to
freezing, drying results in removal of (non-freezable) water normally bound to biomolecules. This is generally more damaging to
biological samples, since the structure and function of biomolecules
are dependent on the presence of liquid water and interactions
therewith. DNA and structural proteins like collagen are relatively
resistant to drying and can often be dried without adverse effects.
Some proteins, however, may suffer from irreversible changes in
their structure upon drying resulting in malfunctioning after rehydration [33]. Liposomal systems cannot be dried without protective measures. Liposomes and lipid bilayers tend to fuse during
drying and undergo lyotropic membrane phase changes resulting
in leakage of aqueous contents across the bilayer [34, 35].
The extent of damage associated with drying of biological
materials increases with sample size and complexity. In a cellular
environment, biomolecules are much more sensitive to oxidative
damage, because reactive oxygen species (ROS) and associated
damage accumulate during drying and subsequent storage. Moreover, anti-oxidative defense systems are compromised at low water
contents, creating opportunities for ROS to react with biomolecules. Free radical-induced injury becomes apparent as lipid peroxidation and phospholipid de-esterification [36–38], DNA
breakage, and accumulation of carbonyl derivatives in proteins
[39]. Furthermore, proteins may be involved in Amadori and
Maillard (i.e., browning) reactions with reducing sugars, particularly at low water contents [40, 41]. Proteins may also be degraded
by proteases originating from lysosomes that lost membrane integrity during dehydration.
8
Willem F. Wolkers and Harrie ¨ tte Oldenhof
results in movement of intracellular water to the extracellular
phase, which causes cells to dehydrate. In addition to restoring
the osmotic equilibrium, this increases the concentration of intracellular molecules and constituents and brings organelles in close
proximity to each other. Solution effects injury refers to cell damage
due to cells being situated in a freeze-concentrated solution of
residual non-freezable water and salts [28, 29]. Cells can tolerate
freezing-induced dehydration to a considerable extent, but if certain tolerance limits are exceeded, viability is lost [26, 27].
Biomolecules (proteins, lipids, DNA, and RNA) may undergo
conformational and phase changes as a consequence of lowering
the temperature and freezing-induced dehydration. Especially
membrane phase changes and changes in the organization of membrane domains during cooling and freezing may cause malfunctioning after reconstitution [30, 31]. Macromolecular assemblies such
as liposomes have been described to suffer from fusion and leakage
during freezing [32].
3.2 Drying Injury
Freezing and drying are different stress factors [21]. In contrast to
freezing, drying results in removal of (non-freezable) water normally bound to biomolecules. This is generally more damaging to
biological samples, since the structure and function of biomolecules
are dependent on the presence of liquid water and interactions
therewith. DNA and structural proteins like collagen are relatively
resistant to drying and can often be dried without adverse effects.
Some proteins, however, may suffer from irreversible changes in
their structure upon drying resulting in malfunctioning after rehydration [33]. Liposomal systems cannot be dried without protective measures. Liposomes and lipid bilayers tend to fuse during
drying and undergo lyotropic membrane phase changes resulting
in leakage of aqueous contents across the bilayer [34, 35].
The extent of damage associated with drying of biological
materials increases with sample size and complexity. In a cellular
environment, biomolecules are much more sensitive to oxidative
damage, because reactive oxygen species (ROS) and associated
damage accumulate during drying and subsequent storage. Moreover, anti-oxidative defense systems are compromised at low water
contents, creating opportunities for ROS to react with biomolecules. Free radical-induced injury becomes apparent as lipid peroxidation and phospholipid de-esterification [36–38], DNA
breakage, and accumulation of carbonyl derivatives in proteins
[39]. Furthermore, proteins may be involved in Amadori and
Maillard (i.e., browning) reactions with reducing sugars, particularly at low water contents [40, 41]. Proteins may also be degraded
by proteases originating from lysosomes that lost membrane integrity during dehydration.
8
Willem F. Wolkers and Harrie ¨ tte Oldenhof