cryoprotectants such as sucrose can prevent protein denaturation
during freezing [391], while glycerol, for example, has been shown
to prevent enzymes such as glucose-6-phosphate dehydrogenase,
carbamoyl phosphate synthetase, and pyruvate decarboxylase from
dissociating into subunits due to cooling [390].
So far, such observations have not been applied to events taking
place in living cells. Perhaps, in the future, an alliance between
cryoenzymologists and cryobiologists could lead to interesting
and potentially utilitarian results. By the same token, there may be
lessons to be learned from those who freeze proteins either in
purified form or in situ [80, 394].
4 Summary and Conclusions
The success of biological vitrification is based on the resolution of a
paradox: most life requires water to survive, and yet the preservation of life by vitrification mostly requires major water depletion.
To avoid the latter, vitrification was attempted first by ultrarapid
cooling and warming and then by rapid cooling and warming in the
presence of the lowest possible concentrations of cryoprotectants,
but over time, it became increasingly apparent that massive replacement of water by chemical agents can in fact be tolerated if carried
out under the right conditions. Today, after decades of successful
empirical investigation of a multitude of different methods of vitrification, elucidation of the mechanisms of biological injury and
protection associated with water depletion is beginning to point
the way toward successful augmentation of the already impressive
ability of complex living systems, including even whole mammalian
organs, to tolerate water replacement to the degree necessary for
safe vitrification and rewarming. These advances are now being
combined with increasingly sophisticated chemical engineering
approaches to the control of ice physics via direct interference
with ice nucleation, growth, and recrystallization, providing even
more expanded opportunities. In addition, new physical techniques
such as laser warming, optimized RF warming, and magnetic
nanoparticle-based warming are adding even more power to vitrification technology and are further ensuring that one of the greatest
current obstacles, devitrification-related injury, can be controlled
even in vitrified whole organs. The use of vitrification to preserve
living systems and the constituents thereof is currently growing at
an exponential rate, and the occurrence of vitrification in organisms
in the natural world is becoming increasingly appreciated. Successful applications have been reported for a wide variety of mammalian
cells and tissues and even for an intact mammalian kidney but
extend as well to the plant kingdom and to the preservation of
insects and other invertebrates of scientific, medical, and commercial interest. And all of this has been accomplished in most cases
without full optimization of the large number of procedural
82
Gregory M. Fahy and Brian Wowk
during freezing [391], while glycerol, for example, has been shown
to prevent enzymes such as glucose-6-phosphate dehydrogenase,
carbamoyl phosphate synthetase, and pyruvate decarboxylase from
dissociating into subunits due to cooling [390].
So far, such observations have not been applied to events taking
place in living cells. Perhaps, in the future, an alliance between
cryoenzymologists and cryobiologists could lead to interesting
and potentially utilitarian results. By the same token, there may be
lessons to be learned from those who freeze proteins either in
purified form or in situ [80, 394].
4 Summary and Conclusions
The success of biological vitrification is based on the resolution of a
paradox: most life requires water to survive, and yet the preservation of life by vitrification mostly requires major water depletion.
To avoid the latter, vitrification was attempted first by ultrarapid
cooling and warming and then by rapid cooling and warming in the
presence of the lowest possible concentrations of cryoprotectants,
but over time, it became increasingly apparent that massive replacement of water by chemical agents can in fact be tolerated if carried
out under the right conditions. Today, after decades of successful
empirical investigation of a multitude of different methods of vitrification, elucidation of the mechanisms of biological injury and
protection associated with water depletion is beginning to point
the way toward successful augmentation of the already impressive
ability of complex living systems, including even whole mammalian
organs, to tolerate water replacement to the degree necessary for
safe vitrification and rewarming. These advances are now being
combined with increasingly sophisticated chemical engineering
approaches to the control of ice physics via direct interference
with ice nucleation, growth, and recrystallization, providing even
more expanded opportunities. In addition, new physical techniques
such as laser warming, optimized RF warming, and magnetic
nanoparticle-based warming are adding even more power to vitrification technology and are further ensuring that one of the greatest
current obstacles, devitrification-related injury, can be controlled
even in vitrified whole organs. The use of vitrification to preserve
living systems and the constituents thereof is currently growing at
an exponential rate, and the occurrence of vitrification in organisms
in the natural world is becoming increasingly appreciated. Successful applications have been reported for a wide variety of mammalian
cells and tissues and even for an intact mammalian kidney but
extend as well to the plant kingdom and to the preservation of
insects and other invertebrates of scientific, medical, and commercial interest. And all of this has been accomplished in most cases
without full optimization of the large number of procedural
82
Gregory M. Fahy and Brian Wowk