slice model, and many examples of the use of npCPAs were
presented [105].
npCPAs, in addition to partially replacing more potentially
toxic pCPA, also tend to increase the viscosity of the solution.
The increase in viscosity increases glass-forming tendency but may
become counterproductive when pCPA diffusion rates need to be
maximized or, particularly, when pCPAs have to be introduced by
perfusion [22].
npCPAs, and particularly lower molecular weight npCPAs,
which are more osmotically active, can also significantly increase
the tonicity of the CPA solution [23]. This is theoretically beneficial
due to the resulting cell shrinkage, which concentrates intracellular
solutes and contributes to intracellular vitrification tendency. Thus,
the npCPA may not just allow intracellular and extracellular glassforming tendencies to be equal at a lower pCPA concentration;
they may actually cause the intracellular glass-forming tendency to
exceed the extracellular glass-forming tendency. If so, this would
tend to offset any danger of IIF associated with vigorous use of
extracellular ice-active agents as discussed above. In addition, cell
shrinkage lowers the absolute amount of pCPA inside the cell,
which enables faster CPA washout after rewarming. However, if
the living system to be preserved is sensitive to chilling injury and
chilling injury is a function of the tonicity of the medium [23] (see
Subheading 3.9), using too much npCPA may exacerbate chilling
injury.
Aside from these caveats, in principle the amount of npCPA
that can be used is limited only by the upper osmotic limit of the cell
in question, the extent to which pCPA levels can be reduced
without compromising the vitrification tendency of the VS, and
the extent to which the npCPA may induce specific toxic effects
[248, 309] (see also below). Although npCPAs do not directly
perturb cytosolic proteins, interactions with the external leaflet of
the plasma membrane and with integral membrane proteins that
communicate with the cytoplasm are still possible. In principle, the
amount of npCPA that can be used might be increased by reducing
carrier solution solutes in favor of more npCPA should cell shrinkage be the limiting factor.
A study by Shaw et al. [310] brought out many important
facets to the use of npCPAs that should be kept in mind. Excessive
amounts of high molecular mass npCPAs tend to raise the T m of the
vitrification solution and increase the total solute concentration
needed to vitrify (both potentially adverse effects) because of the
higher molecular masses and therefore the smaller colligative effects
of npCPAs compared to pCPAs, and npCPAs in a VS have minimal
effects on T G . However, T d (the temperature of devitrification)
tended to be higher with npCPA use, which is beneficial.
70
Gregory M. Fahy and Brian Wowk
presented [105].
npCPAs, in addition to partially replacing more potentially
toxic pCPA, also tend to increase the viscosity of the solution.
The increase in viscosity increases glass-forming tendency but may
become counterproductive when pCPA diffusion rates need to be
maximized or, particularly, when pCPAs have to be introduced by
perfusion [22].
npCPAs, and particularly lower molecular weight npCPAs,
which are more osmotically active, can also significantly increase
the tonicity of the CPA solution [23]. This is theoretically beneficial
due to the resulting cell shrinkage, which concentrates intracellular
solutes and contributes to intracellular vitrification tendency. Thus,
the npCPA may not just allow intracellular and extracellular glassforming tendencies to be equal at a lower pCPA concentration;
they may actually cause the intracellular glass-forming tendency to
exceed the extracellular glass-forming tendency. If so, this would
tend to offset any danger of IIF associated with vigorous use of
extracellular ice-active agents as discussed above. In addition, cell
shrinkage lowers the absolute amount of pCPA inside the cell,
which enables faster CPA washout after rewarming. However, if
the living system to be preserved is sensitive to chilling injury and
chilling injury is a function of the tonicity of the medium [23] (see
Subheading 3.9), using too much npCPA may exacerbate chilling
injury.
Aside from these caveats, in principle the amount of npCPA
that can be used is limited only by the upper osmotic limit of the cell
in question, the extent to which pCPA levels can be reduced
without compromising the vitrification tendency of the VS, and
the extent to which the npCPA may induce specific toxic effects
[248, 309] (see also below). Although npCPAs do not directly
perturb cytosolic proteins, interactions with the external leaflet of
the plasma membrane and with integral membrane proteins that
communicate with the cytoplasm are still possible. In principle, the
amount of npCPA that can be used might be increased by reducing
carrier solution solutes in favor of more npCPA should cell shrinkage be the limiting factor.
A study by Shaw et al. [310] brought out many important
facets to the use of npCPAs that should be kept in mind. Excessive
amounts of high molecular mass npCPAs tend to raise the T m of the
vitrification solution and increase the total solute concentration
needed to vitrify (both potentially adverse effects) because of the
higher molecular masses and therefore the smaller colligative effects
of npCPAs compared to pCPAs, and npCPAs in a VS have minimal
effects on T G . However, T d (the temperature of devitrification)
tended to be higher with npCPA use, which is beneficial.
70
Gregory M. Fahy and Brian Wowk