them from growing even when the temperature is lowered below
the thermodynamic melting point of the ice [46, 55]. Although
antifreeze proteins, or AFPs, were the first natural examples of ice
blockers, ice blockers are usually thought of as being lower in mass
and either synthetic or non-proteinaceous natural products.
Closely related to “ice blockers” are molecules whose primary
utility is their ability to inhibit recrystallization [56]. These “ice
recrystallization inhibitors,” or IRIs, have a strong ability to protect
cells from freezing injury [57, 58] and hold promise for inhibiting
recrystallization after vitrification [59].
A “vitrification solution” [60] is a solution of cryoprotectants
sufficiently concentrated to enable extracellular and intracellular
vitrification of the system at hand under the intended cooling
conditions.
A “carrier solution” is the physiological support medium in
which CPAs are dissolved to enable cells to be exposed to CPAs
without injury beyond the injury associated with the CPAs
themselves.
“Chilling injury” [61] is injury caused by cooling per
se. Although chilling injury is most conspicuous in the absence of
ice, there is strong evidence that it can occur also during freezing in
specific cases [62–66]. “Thermal shock” [67, 68] or “cold shock” is
injury caused by rapid cooling but not by slow cooling, whereas
chilling injury is observed during slow cooling and may even be
“outrun” by very rapid cooling if the system is not subject to injury
from thermal shock.
“Anhydrobiosis” [69, 70] is the survival of life in a desiccated
state. It is relevant to vitrification in the sense that sufficient drying
can concentrate cytoplasm enough to induce a glass transition even
at ambient temperatures, facilitating the prolonged survival of some
organisms and seeds in a dry state [71, 72]. Although it is of
considerable ecological significance and has industrial applications
[71–74], including for therapeutic protein preservation, the present chapter focuses primarily on low temperature vitrification.
1.3 Vitrification
and Molecular Stability
at Low Temperatures
Vitrification is important for protecting cells and tissues against
freezing damage, but it is not as important for preserving the
basic molecular inventory of cells and tissues. Most molecular constituents of cells are reasonably stable under low temperature conditions in situ even without special precautions, although there are
exceptions. Generally speaking, neither freezing and thawing nor
cooling per se causes the formation or breakage of covalent chemical bonds. The reversible formation of S–S cross-links in frozen
thiogels [75], one particular protein (but not others) extracted
from freeze-killed cabbage [76], and one of five SH groups in
F-actin [77] has been reported, but no change in S–S or S–H
content was found in lethally frozen sea urchin eggs [78], and an
increase of S–H content in frozen-thawed bull spermatozoon
32
Gregory M. Fahy and Brian Wowk
the thermodynamic melting point of the ice [46, 55]. Although
antifreeze proteins, or AFPs, were the first natural examples of ice
blockers, ice blockers are usually thought of as being lower in mass
and either synthetic or non-proteinaceous natural products.
Closely related to “ice blockers” are molecules whose primary
utility is their ability to inhibit recrystallization [56]. These “ice
recrystallization inhibitors,” or IRIs, have a strong ability to protect
cells from freezing injury [57, 58] and hold promise for inhibiting
recrystallization after vitrification [59].
A “vitrification solution” [60] is a solution of cryoprotectants
sufficiently concentrated to enable extracellular and intracellular
vitrification of the system at hand under the intended cooling
conditions.
A “carrier solution” is the physiological support medium in
which CPAs are dissolved to enable cells to be exposed to CPAs
without injury beyond the injury associated with the CPAs
themselves.
“Chilling injury” [61] is injury caused by cooling per
se. Although chilling injury is most conspicuous in the absence of
ice, there is strong evidence that it can occur also during freezing in
specific cases [62–66]. “Thermal shock” [67, 68] or “cold shock” is
injury caused by rapid cooling but not by slow cooling, whereas
chilling injury is observed during slow cooling and may even be
“outrun” by very rapid cooling if the system is not subject to injury
from thermal shock.
“Anhydrobiosis” [69, 70] is the survival of life in a desiccated
state. It is relevant to vitrification in the sense that sufficient drying
can concentrate cytoplasm enough to induce a glass transition even
at ambient temperatures, facilitating the prolonged survival of some
organisms and seeds in a dry state [71, 72]. Although it is of
considerable ecological significance and has industrial applications
[71–74], including for therapeutic protein preservation, the present chapter focuses primarily on low temperature vitrification.
1.3 Vitrification
and Molecular Stability
at Low Temperatures
Vitrification is important for protecting cells and tissues against
freezing damage, but it is not as important for preserving the
basic molecular inventory of cells and tissues. Most molecular constituents of cells are reasonably stable under low temperature conditions in situ even without special precautions, although there are
exceptions. Generally speaking, neither freezing and thawing nor
cooling per se causes the formation or breakage of covalent chemical bonds. The reversible formation of S–S cross-links in frozen
thiogels [75], one particular protein (but not others) extracted
from freeze-killed cabbage [76], and one of five SH groups in
F-actin [77] has been reported, but no change in S–S or S–H
content was found in lethally frozen sea urchin eggs [78], and an
increase of S–H content in frozen-thawed bull spermatozoon
32
Gregory M. Fahy and Brian Wowk