The ability of ice to form during either cooling or warming
depends on how much time is available for ice nucleation and
growth. Therefore, at sufficiently high cooling and warming rates,
it is often possible to “outrun” the kinetics of ice formation sufficiently to enable survival. The “critical cooling rate” is the cooling
rate above which appreciable ice formation is not observed [41],
and the “critical warming rate” is the warming rate that completely
or sufficiently suppresses ice formation during warming [41]. The
critical cooling [42] and warming [43] rates for a given system
depend very strongly on the total solute content of the system
and also on the chemical nature of the solute.
The solutes used for vitrification are generally the same as or
similar to those used to protect against freezing injury and are
generally referred to as “cryoprotective agents” (CPAs) or “cryoprotectants” [44–46]. CPAs that are of sufficiently low molecular
mass to pass reasonably quickly across cell membranes are referred
to as “penetrating” or “permeating” CPAs (pCPAs), while those of
higher molecular mass are referred to as “non-penetrating” CPAs
(npCPAs). Glycerol, which has a molecular mass of 92.1 Da, is near
the limit for defining the difference between pCPAs and npCPAs,
although less hydrophilic CPAs may penetrate despite somewhat
higher total masses due to their greater solubility in membrane
lipids. To be an effective pCPA, the agent must be miscible or
soluble in water to high concentrations, be of low toxicity, be able
to remain in solution even at very low temperatures, and, as noted
above, be able to cross the cell membrane [44, 47]. npCPAs have
similar requirements, but by definition do not enter cells over
practical time spans. pCPAs include dimethyl sulfoxide, ethylene
glycol, glycerol, and propylene glycol, but many others have been
identified [45]. To cross cell membranes, pCPAs must not possess a
net fixed charge, although molecules that are charged only transiently can serve as pCPAs [44]. npCPAs include polyvinylpyrrolidone, polyethylene glycol, sucrose, trehalose, and many others.
Interestingly, trehalose can be converted into a pCPA by
acetylation [48].
It has been argued that when cryoprotectants are used to
enable vitrification they should be called “vitrificants” rather than
“cryoprotectants” [49]. However, cryoprotectants are defined to
be agents that reduce or prevent freezing injury, which they certainly do when they prevent freezing altogether, so the term “cryoprotectants” remains proper in the context of vitrification.
Nonetheless, the term “vitrificants,” while not widely used, is also
correctly descriptive of agents that facilitate vitrification.
A relatively new type of cryoprotectant is the “ice blocker,”
which is a molecule that is capable of undergoing specific interactions with ice or ice-nucleating agents so as to reduce or prevent ice
nucleation, ice growth, or both [19, 50–54]. “Antifreeze proteins”
are proteins that can adsorb to the surface of ice crystals and prevent
Principles of Vitrification
31
depends on how much time is available for ice nucleation and
growth. Therefore, at sufficiently high cooling and warming rates,
it is often possible to “outrun” the kinetics of ice formation sufficiently to enable survival. The “critical cooling rate” is the cooling
rate above which appreciable ice formation is not observed [41],
and the “critical warming rate” is the warming rate that completely
or sufficiently suppresses ice formation during warming [41]. The
critical cooling [42] and warming [43] rates for a given system
depend very strongly on the total solute content of the system
and also on the chemical nature of the solute.
The solutes used for vitrification are generally the same as or
similar to those used to protect against freezing injury and are
generally referred to as “cryoprotective agents” (CPAs) or “cryoprotectants” [44–46]. CPAs that are of sufficiently low molecular
mass to pass reasonably quickly across cell membranes are referred
to as “penetrating” or “permeating” CPAs (pCPAs), while those of
higher molecular mass are referred to as “non-penetrating” CPAs
(npCPAs). Glycerol, which has a molecular mass of 92.1 Da, is near
the limit for defining the difference between pCPAs and npCPAs,
although less hydrophilic CPAs may penetrate despite somewhat
higher total masses due to their greater solubility in membrane
lipids. To be an effective pCPA, the agent must be miscible or
soluble in water to high concentrations, be of low toxicity, be able
to remain in solution even at very low temperatures, and, as noted
above, be able to cross the cell membrane [44, 47]. npCPAs have
similar requirements, but by definition do not enter cells over
practical time spans. pCPAs include dimethyl sulfoxide, ethylene
glycol, glycerol, and propylene glycol, but many others have been
identified [45]. To cross cell membranes, pCPAs must not possess a
net fixed charge, although molecules that are charged only transiently can serve as pCPAs [44]. npCPAs include polyvinylpyrrolidone, polyethylene glycol, sucrose, trehalose, and many others.
Interestingly, trehalose can be converted into a pCPA by
acetylation [48].
It has been argued that when cryoprotectants are used to
enable vitrification they should be called “vitrificants” rather than
“cryoprotectants” [49]. However, cryoprotectants are defined to
be agents that reduce or prevent freezing injury, which they certainly do when they prevent freezing altogether, so the term “cryoprotectants” remains proper in the context of vitrification.
Nonetheless, the term “vitrificants,” while not widely used, is also
correctly descriptive of agents that facilitate vitrification.
A relatively new type of cryoprotectant is the “ice blocker,”
which is a molecule that is capable of undergoing specific interactions with ice or ice-nucleating agents so as to reduce or prevent ice
nucleation, ice growth, or both [19, 50–54]. “Antifreeze proteins”
are proteins that can adsorb to the surface of ice crystals and prevent
Principles of Vitrification
31