(polar groups) present on all of the pCPAs in that same solution. As
glass-forming tendency increases, the water content of the solution
at its C V rises relative to the number of chemical groups available to
prevent water from freezing because the average polar group in the
solution is able to interact with and prevent the freezing of more
water molecules owing to its stronger hydrogen bonding with
water, and qv∗ therefore increases. Viability concomitantly
declines, suggesting a harmful competition between stronger
glass-forming chemical groups and vital cellular biomolecules for
access to water. The principle of favoring weak glass formers for the
formulation of vitrification solutions (VSs) has enabled the development of less toxic VSs [23, 245], but there are limits on its
application. For example, the weak glass former, ethylene glycol,
may have specific toxic effects at higher concentrations [245] or
may be required in such high concentrations so as to begin to
deplete water to the point where water becomes inadequately
available despite weak interactions with the pCPA. Glycerol,
which is another weak glass former, is frequently too impermeable,
too viscous, or too toxic for other reasons (perhaps including its
ability to be phosphorylated at the expense of cellular ATP stores
[307, 308]) to be used in concentrations that might be more ideal
in theory. Despite such limitations, however, favoring weak glass
formers has, in combination with other modalities discussed above,
enabled the creation of M22, a 9.4 M solution whose critical cooling and warming rates are extremely low, yet which can be perfused
through a kidney with only transient dysfunction after
transplantation [23].
3.6 Extracellular
Agents in Vitrification
The physical and biological utility of ice-active extracellular agents
is discussed in Subheadings 2.6 and 3.5. The present discussion is
confined to conventional npCPAs.
As noted in Subheading 3.1, living cells contain significant
amounts of protein, metabolic intermediates, nuclei acids, and
other solutes. These solutes might, when water content is significantly reduced by the use of pCPAs, contribute more to glassforming tendency of the cytosol than the carrier solution contributes to the stability of the extracellular solution. In this case, the
use of pCPA alone to ensure vitrification of the extracellular solution would actually require more pCPA than needed to vitrify the
intracellular solutions and would therefore be more toxic than
necessary. To correct this problem, it was suggested in 1981
[131] and reported in 1982 [132] that extracellular pCPA concentrations could be reduced by using npCPAs as counterparts to
intracellular solutes, thus maintaining the vitrification tendency of
the vitrification solution with less pCPA while still allowing cells in
contact with this solution to vitrify. This was later documented by
the visual appearance or lack of appearance of ice in a rabbit kidney
Principles of Vitrification
69
glass-forming tendency increases, the water content of the solution
at its C V rises relative to the number of chemical groups available to
prevent water from freezing because the average polar group in the
solution is able to interact with and prevent the freezing of more
water molecules owing to its stronger hydrogen bonding with
water, and qv∗ therefore increases. Viability concomitantly
declines, suggesting a harmful competition between stronger
glass-forming chemical groups and vital cellular biomolecules for
access to water. The principle of favoring weak glass formers for the
formulation of vitrification solutions (VSs) has enabled the development of less toxic VSs [23, 245], but there are limits on its
application. For example, the weak glass former, ethylene glycol,
may have specific toxic effects at higher concentrations [245] or
may be required in such high concentrations so as to begin to
deplete water to the point where water becomes inadequately
available despite weak interactions with the pCPA. Glycerol,
which is another weak glass former, is frequently too impermeable,
too viscous, or too toxic for other reasons (perhaps including its
ability to be phosphorylated at the expense of cellular ATP stores
[307, 308]) to be used in concentrations that might be more ideal
in theory. Despite such limitations, however, favoring weak glass
formers has, in combination with other modalities discussed above,
enabled the creation of M22, a 9.4 M solution whose critical cooling and warming rates are extremely low, yet which can be perfused
through a kidney with only transient dysfunction after
transplantation [23].
3.6 Extracellular
Agents in Vitrification
The physical and biological utility of ice-active extracellular agents
is discussed in Subheadings 2.6 and 3.5. The present discussion is
confined to conventional npCPAs.
As noted in Subheading 3.1, living cells contain significant
amounts of protein, metabolic intermediates, nuclei acids, and
other solutes. These solutes might, when water content is significantly reduced by the use of pCPAs, contribute more to glassforming tendency of the cytosol than the carrier solution contributes to the stability of the extracellular solution. In this case, the
use of pCPA alone to ensure vitrification of the extracellular solution would actually require more pCPA than needed to vitrify the
intracellular solutions and would therefore be more toxic than
necessary. To correct this problem, it was suggested in 1981
[131] and reported in 1982 [132] that extracellular pCPA concentrations could be reduced by using npCPAs as counterparts to
intracellular solutes, thus maintaining the vitrification tendency of
the vitrification solution with less pCPA while still allowing cells in
contact with this solution to vitrify. This was later documented by
the visual appearance or lack of appearance of ice in a rabbit kidney
Principles of Vitrification
69