Society for Cryobiology that when hearts loaded with EG were
transferred into liquid nitrogen and then rapidly rewarmed, they
“shattered,” as he put it (a general problem that is discussed in
detail below). He later reported that 10 M EG was the minimum
concentration allowing recovery of frog hearts from À79
C, but
that rat hearts could not tolerate more than 5 M EG and therefore
could not be successfully preserved [118]. Nevertheless, establishing that frog hearts, at least, can theoretically be vitrified and
recovered remains one of the most outstanding achievements on
the path toward biological vitrification.
From 1970 [119] to 1972 [120], Elford similarly worked out a
method for preserving strips of intestinal smooth muscle in a supercooled state at À79
C using variations of Farrant’s method. In the
1972 work, although it was not reported formally, it was noted
anecdotally (D.E. Pegg, personal communication) that some muscle strips cooled in liquid nitrogen (the ones that had not experienced the same kind of “shattering” or fracturing observed by
Rapatz) recovered after warming and would therefore have been
the first successfully and definitively vitrified organized tissues.
However, once again, no such observations were reported, and
there was no suggestion that vitrification as opposed to deep supercooling might be used as a method of cryopreservation.
1.4.3
Cryoprotectant-Enabled
Vitrification: 1977–1986
A turning point in the history of vitrification was achieved in a
landmark paper by Pierre Boutron in 1978 [41]. Boutron, a physicist interested in amorphous solid water [121] and inspired in part
by the work of Luyet and colleagues on the vitrification of aqueous
cryoprotectant solutions, was the first to explicitly state that “in the
extreme case of a solution which remains entirely amorphous even
at very slow cooling or warming rates, all cells should be protected.” This concept differed from Luyet’s classical approach in
that it recognized that mixing water with cryoprotectants inhibits
crystallization (stabilizes the amorphous state) and therefore
reduces the cooling rate needed for vitrification. This is the essential
basis of virtually all modern methods of vitrification. However,
Boutron tacitly assumed (quite reasonably) that such a high degree
of stability could not be achieved without lethal cryoprotectant
toxicity and therefore emphasized the fact that if cells were treated
with much higher than conventional concentrations of cryoprotectant, they might be induced to vitrify at cooling rates that were
much higher than usual, but still much lower than what would be
demanded by Luyet’s approach.
Boutron was the first to thoroughly describe the kinetics of ice
formation in vitrifiable aqueous cryoprotectant solutions, to combine the use of X-ray diffraction and differential scanning calorimetry (DSC) to investigate ice formation and glass transitions in
aqueous solutions, to describe the concepts of the critical cooling
36
Gregory M. Fahy and Brian Wowk
transferred into liquid nitrogen and then rapidly rewarmed, they
“shattered,” as he put it (a general problem that is discussed in
detail below). He later reported that 10 M EG was the minimum
concentration allowing recovery of frog hearts from À79
C, but
that rat hearts could not tolerate more than 5 M EG and therefore
could not be successfully preserved [118]. Nevertheless, establishing that frog hearts, at least, can theoretically be vitrified and
recovered remains one of the most outstanding achievements on
the path toward biological vitrification.
From 1970 [119] to 1972 [120], Elford similarly worked out a
method for preserving strips of intestinal smooth muscle in a supercooled state at À79
C using variations of Farrant’s method. In the
1972 work, although it was not reported formally, it was noted
anecdotally (D.E. Pegg, personal communication) that some muscle strips cooled in liquid nitrogen (the ones that had not experienced the same kind of “shattering” or fracturing observed by
Rapatz) recovered after warming and would therefore have been
the first successfully and definitively vitrified organized tissues.
However, once again, no such observations were reported, and
there was no suggestion that vitrification as opposed to deep supercooling might be used as a method of cryopreservation.
1.4.3
Cryoprotectant-Enabled
Vitrification: 1977–1986
A turning point in the history of vitrification was achieved in a
landmark paper by Pierre Boutron in 1978 [41]. Boutron, a physicist interested in amorphous solid water [121] and inspired in part
by the work of Luyet and colleagues on the vitrification of aqueous
cryoprotectant solutions, was the first to explicitly state that “in the
extreme case of a solution which remains entirely amorphous even
at very slow cooling or warming rates, all cells should be protected.” This concept differed from Luyet’s classical approach in
that it recognized that mixing water with cryoprotectants inhibits
crystallization (stabilizes the amorphous state) and therefore
reduces the cooling rate needed for vitrification. This is the essential
basis of virtually all modern methods of vitrification. However,
Boutron tacitly assumed (quite reasonably) that such a high degree
of stability could not be achieved without lethal cryoprotectant
toxicity and therefore emphasized the fact that if cells were treated
with much higher than conventional concentrations of cryoprotectant, they might be induced to vitrify at cooling rates that were
much higher than usual, but still much lower than what would be
demanded by Luyet’s approach.
Boutron was the first to thoroughly describe the kinetics of ice
formation in vitrifiable aqueous cryoprotectant solutions, to combine the use of X-ray diffraction and differential scanning calorimetry (DSC) to investigate ice formation and glass transitions in
aqueous solutions, to describe the concepts of the critical cooling
36
Gregory M. Fahy and Brian Wowk