rate and the critical warming rate, and to introduce mathematical
models of the kinetics of ice formation relevant to vitrifiable solutions [41]. However, his goal was to find solutions that would
vitrify at low and therefore nontoxic cryoprotectant concentrations,
but he also showed that even rather concentrated solutions have
impossibly high critical warming rates (e.g., the critical warming
rate of 45% w/w glycerol is about 3 Â 10
11 C/min) [41], suggesting that his approach would not be feasible in practice. Nevertheless, Boutron and Kaufmann went on to study, in 1978–1979, the
stability of the amorphous state of many aqueous cryoprotectant
solutions [122–124], most significantly including propylene glycol
(PG, or 1,2-propanediol) [125]. Remarkably, 35% w/w PG could
be vitrified when cooled at only 320
C/min, and 40% PG vitrified
when cooled at ~40
C/min. The critical warming rate for 45%
w/w PG was a remarkably low 260
C/min, but this was still a very
daunting concentration for the recovery of cellular viability, and the
critical warming rate for 40% w/w PG was extrapolated to be
76,000
C/min.
In 1977, James and Farrant observed that small population
fractions of the parasitic worm, Schistosoma mansoni, were able to
survive after slow freezing to À30
C in methanol followed by
plunging into liquid nitrogen and reasoned that the 40% concentration of methanol induced by slow freezing would likely vitrify
upon further abrupt cooling [126]. Following up on this observation, James observed, in 1980, that it should be possible to obtain
the same result “more simply by using a high cryoprotectant concentration and rapid cooling. The initial slow cooling step could
thus be omitted and the chance of damaging effects of extra and
intracellular ice formation eliminated” [127]. This method met
with some success, but evidence suggested that the worms were
in fact not taking up the methanol and therefore were merely
surviving rapid freezing.
After investigation of the feasibility of whole organ deep supercooling in 1977–1980 [88, 89, 128], Fahy proposed, in
1981–1984 [105, 129–134], a different approach to vitrification:
the extension of deep supercooling all the way down to T G . This
method, motivated by the need to prevent mechanical injury from
ice in whole organs [18, 60, 89, 105, 129], relies on the fact that at
sufficiently high concentrations, both critical cooling rates and
critical warming rates become low enough to enable, in principle,
even the vitrification of objects as large as human organs. It introduced a new problem, however, the toxicity of very high concentrations of cryoprotectants. For large organs, these problems
cannot be circumvented by rapid cooling and warming or by exposure to these agents at temperatures as low as À55
C as in the
methods of Farrant, Rapatz, and Elford due to the fact that vascularized organs must be protected by perfusion, and perfusion rates
Principles of Vitrification
37
models of the kinetics of ice formation relevant to vitrifiable solutions [41]. However, his goal was to find solutions that would
vitrify at low and therefore nontoxic cryoprotectant concentrations,
but he also showed that even rather concentrated solutions have
impossibly high critical warming rates (e.g., the critical warming
rate of 45% w/w glycerol is about 3 Â 10
11 C/min) [41], suggesting that his approach would not be feasible in practice. Nevertheless, Boutron and Kaufmann went on to study, in 1978–1979, the
stability of the amorphous state of many aqueous cryoprotectant
solutions [122–124], most significantly including propylene glycol
(PG, or 1,2-propanediol) [125]. Remarkably, 35% w/w PG could
be vitrified when cooled at only 320
C/min, and 40% PG vitrified
when cooled at ~40
C/min. The critical warming rate for 45%
w/w PG was a remarkably low 260
C/min, but this was still a very
daunting concentration for the recovery of cellular viability, and the
critical warming rate for 40% w/w PG was extrapolated to be
76,000
C/min.
In 1977, James and Farrant observed that small population
fractions of the parasitic worm, Schistosoma mansoni, were able to
survive after slow freezing to À30
C in methanol followed by
plunging into liquid nitrogen and reasoned that the 40% concentration of methanol induced by slow freezing would likely vitrify
upon further abrupt cooling [126]. Following up on this observation, James observed, in 1980, that it should be possible to obtain
the same result “more simply by using a high cryoprotectant concentration and rapid cooling. The initial slow cooling step could
thus be omitted and the chance of damaging effects of extra and
intracellular ice formation eliminated” [127]. This method met
with some success, but evidence suggested that the worms were
in fact not taking up the methanol and therefore were merely
surviving rapid freezing.
After investigation of the feasibility of whole organ deep supercooling in 1977–1980 [88, 89, 128], Fahy proposed, in
1981–1984 [105, 129–134], a different approach to vitrification:
the extension of deep supercooling all the way down to T G . This
method, motivated by the need to prevent mechanical injury from
ice in whole organs [18, 60, 89, 105, 129], relies on the fact that at
sufficiently high concentrations, both critical cooling rates and
critical warming rates become low enough to enable, in principle,
even the vitrification of objects as large as human organs. It introduced a new problem, however, the toxicity of very high concentrations of cryoprotectants. For large organs, these problems
cannot be circumvented by rapid cooling and warming or by exposure to these agents at temperatures as low as À55
C as in the
methods of Farrant, Rapatz, and Elford due to the fact that vascularized organs must be protected by perfusion, and perfusion rates
Principles of Vitrification
37