being demonstrated, and alteration of the heat shock response or
other biochemical pathways are approaches that presently remain
mostly theoretical and are discussed in Subheading 3.8, so further
elaboration here on the last seven approaches to toxicity mitigation
will be confined to comments on the use of ice blockers and
methoxylated CPAs, cost function optimization, and preferential
use of weak glass formers.
As noted above, ice blockers interact with, and inhibit, extracellular ice and thereby find utility in part by reducing the amount
of pCPA otherwise needed and lowering toxicity in this way.
This advantage is possible despite the fact that established
ice-blocking molecules do not have access to the intracellular
space because most cells do not appear to contain heterogeneous
nucleating agents of any significant effectiveness. Evidence for this
conclusion comes from a variety of observations (see, e.g., Table 1
of [39] and its accompanying discussion) but most directly from
experiments in which single cells were supercooled to temperatures
near T h and found to have minimal nucleating activity
[206, 305]. Even in the case of the mammalian kidney, for which
devitrification in the inner medulla appears to originate intracellularly [219], the origin of devitrification is apparently not intracellular nucleators, but slightly inadequate cellular uptake of pCPAs
[236], although the lack of intracellular ice blocker might in principle also play a role.
The latter observation shows that it is possible for unusual
situations to exist in which the extracellular medium is more resistant to ice formation than is the intracellular compartment. Given
this, excessive reliance on extracellular ice growth inhibition, either
achieved by using ice blockers or by using high molecular weight
viscosity-enhancing agents, might make the extracellular medium
more resistant to ice nucleation or growth than the cytoplasm, thus
potentially permitting intracellular ice formation not predicted
from the behavior of the medium. In part for this reason, caution
is presently suggested in the use of ice-blocking agents to reduce
pCPA concentrations in order to lower toxicity. In the future, the
use of IRIs that can penetrate cells [57] may ease this restriction
both in normal cells and in cells like those of the renal inner medulla
to the degree that ice recrystallization inhibition is sufficient to
enable survival of intracellular ice formation.
Replacing hydroxyl groups on OH-bearing pCPAs with methoxy groups has two favorable effects: glass-forming tendency is
greatly improved and membrane permeability is greatly increased
[297]. There are, however, two counterbalancing negative effects:
increasing the glass-forming tendency of pCPAs generally increases
toxicity in the context of vitrification (see Subheading 3.8), and
increased membrane permeability is due to increased hydrophobicity, which also tends to make pCPAs more toxic [306]. However,
with at least one methoxylated pCPA, “methoxy-glycerol”
Principles of Vitrification
67
other biochemical pathways are approaches that presently remain
mostly theoretical and are discussed in Subheading 3.8, so further
elaboration here on the last seven approaches to toxicity mitigation
will be confined to comments on the use of ice blockers and
methoxylated CPAs, cost function optimization, and preferential
use of weak glass formers.
As noted above, ice blockers interact with, and inhibit, extracellular ice and thereby find utility in part by reducing the amount
of pCPA otherwise needed and lowering toxicity in this way.
This advantage is possible despite the fact that established
ice-blocking molecules do not have access to the intracellular
space because most cells do not appear to contain heterogeneous
nucleating agents of any significant effectiveness. Evidence for this
conclusion comes from a variety of observations (see, e.g., Table 1
of [39] and its accompanying discussion) but most directly from
experiments in which single cells were supercooled to temperatures
near T h and found to have minimal nucleating activity
[206, 305]. Even in the case of the mammalian kidney, for which
devitrification in the inner medulla appears to originate intracellularly [219], the origin of devitrification is apparently not intracellular nucleators, but slightly inadequate cellular uptake of pCPAs
[236], although the lack of intracellular ice blocker might in principle also play a role.
The latter observation shows that it is possible for unusual
situations to exist in which the extracellular medium is more resistant to ice formation than is the intracellular compartment. Given
this, excessive reliance on extracellular ice growth inhibition, either
achieved by using ice blockers or by using high molecular weight
viscosity-enhancing agents, might make the extracellular medium
more resistant to ice nucleation or growth than the cytoplasm, thus
potentially permitting intracellular ice formation not predicted
from the behavior of the medium. In part for this reason, caution
is presently suggested in the use of ice-blocking agents to reduce
pCPA concentrations in order to lower toxicity. In the future, the
use of IRIs that can penetrate cells [57] may ease this restriction
both in normal cells and in cells like those of the renal inner medulla
to the degree that ice recrystallization inhibition is sufficient to
enable survival of intracellular ice formation.
Replacing hydroxyl groups on OH-bearing pCPAs with methoxy groups has two favorable effects: glass-forming tendency is
greatly improved and membrane permeability is greatly increased
[297]. There are, however, two counterbalancing negative effects:
increasing the glass-forming tendency of pCPAs generally increases
toxicity in the context of vitrification (see Subheading 3.8), and
increased membrane permeability is due to increased hydrophobicity, which also tends to make pCPAs more toxic [306]. However,
with at least one methoxylated pCPA, “methoxy-glycerol”
Principles of Vitrification
67