4 Mode of Action of Protectants
4.1 Cryoprotective
Agents
CPAs are often categorized based on their ability to permeate
membranes. Membrane-permeable CPAs are small polar molecules
that are able to easily pass cellular membranes by passive diffusion.
Membrane-impermeable CPAs can be divided into smaller molecules that are still osmotically active (e.g., sugars) and large (bio)
polymers that have little effects on the medium osmolality and thus
are considered osmotically inactive [42]. Membrane-impermeable
agents are typically used in combination with a membranepermeable CPA that can provide intracellular protection.
One of the most obvious general modes of action of CPAs is the
reduction of the amount of ice formed, simply by increasing the
total solute concentration. This is the case for both membranepermeable and membrane-impermeable CPAs. Early studies
showed that glycerol modulates the rise in salt concentration during freezing, therewith reducing solution effects injury
[28, 29]. Later, it has been postulated that cryoprotective action
is related to the affinity of specific agents to bind with biomolecules
therewith preventing phase and conformational changes. The preferential exclusion theory explains the stabilizing effects of protective agents on proteins from a thermodynamic viewpoint [43]. In a
solution consisting of water, a protein, and, e.g., DMSO, the
protein preferentially interacts with water rather than with
DMSO. In other words, DMSO is preferentially excluded from
the protein surface. This creates a thermodynamically unfavorable
situation, which stabilizes the native over the unfolded protein state
by increasing the free energy barrier for protein unfolding and
denaturation (see Fig. 2a, b). The preferential exclusion theory is
developed for proteins in dilute solutions at normal temperatures,
but it is believed that this theory also explains the stabilizing effect
of CPAs during cooling. Also membranes are stabilized by molecules that are preferentially excluded from the membrane
surface [44].
CPAs also protect by modulating the rate at which a cell dehydrates during freezing, by decreasing the activation energy for
water transport across the membrane [45, 46]. This results in a
more gradual dehydration while minimizing the likelihood of intracellular ice formation. Moreover, CPAs facilitate the formation of a
vitrified state, which aids protection to cells during freezing. When
low CPA concentrations and a slow cooling rate are used, the
so-called maximally freeze-concentrated solution that is formed
during freezing forms a glassy state below a characteristic temperature referred to as T g
0 (see Fig. 1c). When sufficiently high CPA
concentrations and fast enough cooling rates are used, the entire
solution enters into a vitrified state. Whereas T g
0 does not change
with solute concentration, the glass transition temperature, T g , is
Principles of Cryopreservation and Freeze-Drying
9
4.1 Cryoprotective
Agents
CPAs are often categorized based on their ability to permeate
membranes. Membrane-permeable CPAs are small polar molecules
that are able to easily pass cellular membranes by passive diffusion.
Membrane-impermeable CPAs can be divided into smaller molecules that are still osmotically active (e.g., sugars) and large (bio)
polymers that have little effects on the medium osmolality and thus
are considered osmotically inactive [42]. Membrane-impermeable
agents are typically used in combination with a membranepermeable CPA that can provide intracellular protection.
One of the most obvious general modes of action of CPAs is the
reduction of the amount of ice formed, simply by increasing the
total solute concentration. This is the case for both membranepermeable and membrane-impermeable CPAs. Early studies
showed that glycerol modulates the rise in salt concentration during freezing, therewith reducing solution effects injury
[28, 29]. Later, it has been postulated that cryoprotective action
is related to the affinity of specific agents to bind with biomolecules
therewith preventing phase and conformational changes. The preferential exclusion theory explains the stabilizing effects of protective agents on proteins from a thermodynamic viewpoint [43]. In a
solution consisting of water, a protein, and, e.g., DMSO, the
protein preferentially interacts with water rather than with
DMSO. In other words, DMSO is preferentially excluded from
the protein surface. This creates a thermodynamically unfavorable
situation, which stabilizes the native over the unfolded protein state
by increasing the free energy barrier for protein unfolding and
denaturation (see Fig. 2a, b). The preferential exclusion theory is
developed for proteins in dilute solutions at normal temperatures,
but it is believed that this theory also explains the stabilizing effect
of CPAs during cooling. Also membranes are stabilized by molecules that are preferentially excluded from the membrane
surface [44].
CPAs also protect by modulating the rate at which a cell dehydrates during freezing, by decreasing the activation energy for
water transport across the membrane [45, 46]. This results in a
more gradual dehydration while minimizing the likelihood of intracellular ice formation. Moreover, CPAs facilitate the formation of a
vitrified state, which aids protection to cells during freezing. When
low CPA concentrations and a slow cooling rate are used, the
so-called maximally freeze-concentrated solution that is formed
during freezing forms a glassy state below a characteristic temperature referred to as T g
0 (see Fig. 1c). When sufficiently high CPA
concentrations and fast enough cooling rates are used, the entire
solution enters into a vitrified state. Whereas T g
0 does not change
with solute concentration, the glass transition temperature, T g , is
Principles of Cryopreservation and Freeze-Drying
9