gelatin gels might vitrify based on studies from the 1920s. He also
documented his own work corroborating and extending those
observations using gelatin gels, egg albumin, and leaves of various
plant species, finding evidence for at least partial vitrification based
on maintenance of translucency after quenching and opacification
(believed to be devitrification) on warming.
However, Luyet’s seminal paper did not include tests to establish whether living cells could actually survive after being vitrified
and rewarmed. This gap was remedied in dramatic fashion the year
after Luyet’s proposal with an avalanche of studies of cryopreservation by vitrification, now including evidence of cellular survival after
rewarming of diverse cell types. There were at least six papers by
Luyet and colleagues [94] and five communications [95] by Goetz
and Goetz [95, 96], who had their own original thoughts on
theoretical aspects of vitrification as well [96]. One of Luyet’s
1938 papers appeared in Science [97]. Thus, the concept of vitrification as a method of cryopreservation was launched with considerable scientific enthusiasm.
This enthusiasm continued until 1954, when Luyet’s evidence
for vitrification (namely, transparency or translucency upon
quenching or survival after rewarming) was publicly challenged by
Audrey Smith [98]. This led first to a disclaimer acknowledging the
tentative nature of Luyet’s conclusions [99] and then to experiments that ultimately, in 1958, verified the validity of Smith’s
criticisms: rapid freezing can lead to crystals too small or too thin
to scatter visible light, giving the false impression that vitrification
has been achieved [100, 101]. From that point forward, Luyet
never made any further claims of vitrification and ultimately concluded that his efforts had been “mostly negative” and that the
scientific view of vitrification was that it was “mostly academic”
because of its confinement to very small systems that could be
cooled and warmed at ultrarapid rates [102].
1.4.2 Information
Development, 1965–1972:
Supercooling
and Vitrification
Tendencies
of Cryoprotectant-Water
Solutions
Meanwhile, completely independently of Luyet and the goal of
vitrification, John Farrant was seeking ways to preserve organized
tissues without ice crystal damage by using an entirely different
approach, namely, freezing point depression followed by extreme
supercooling [103]. He reported that by introducing 55% v/v
dimethyl sulfoxide (Me 2 SO) in a series of small steps as temperature
was commensurately reduced in a series of small steps—a technique
now referred to as “liquidus tracking” [104]—he could avoid the
toxic effects of this enormous concentration of cryoprotectant and
successfully preserve guinea pig uteri in a supercooled state at
À79
C (the temperature of subliming dry ice) with subsequent
excellent contractile function after rewarming and removal of the
Me 2 SO. This concentration of Me 2 SO is now known to be sufficient for vitrification [105], so Farrant could have actually achieved
34
Gregory M. Fahy and Brian Wowk
documented his own work corroborating and extending those
observations using gelatin gels, egg albumin, and leaves of various
plant species, finding evidence for at least partial vitrification based
on maintenance of translucency after quenching and opacification
(believed to be devitrification) on warming.
However, Luyet’s seminal paper did not include tests to establish whether living cells could actually survive after being vitrified
and rewarmed. This gap was remedied in dramatic fashion the year
after Luyet’s proposal with an avalanche of studies of cryopreservation by vitrification, now including evidence of cellular survival after
rewarming of diverse cell types. There were at least six papers by
Luyet and colleagues [94] and five communications [95] by Goetz
and Goetz [95, 96], who had their own original thoughts on
theoretical aspects of vitrification as well [96]. One of Luyet’s
1938 papers appeared in Science [97]. Thus, the concept of vitrification as a method of cryopreservation was launched with considerable scientific enthusiasm.
This enthusiasm continued until 1954, when Luyet’s evidence
for vitrification (namely, transparency or translucency upon
quenching or survival after rewarming) was publicly challenged by
Audrey Smith [98]. This led first to a disclaimer acknowledging the
tentative nature of Luyet’s conclusions [99] and then to experiments that ultimately, in 1958, verified the validity of Smith’s
criticisms: rapid freezing can lead to crystals too small or too thin
to scatter visible light, giving the false impression that vitrification
has been achieved [100, 101]. From that point forward, Luyet
never made any further claims of vitrification and ultimately concluded that his efforts had been “mostly negative” and that the
scientific view of vitrification was that it was “mostly academic”
because of its confinement to very small systems that could be
cooled and warmed at ultrarapid rates [102].
1.4.2 Information
Development, 1965–1972:
Supercooling
and Vitrification
Tendencies
of Cryoprotectant-Water
Solutions
Meanwhile, completely independently of Luyet and the goal of
vitrification, John Farrant was seeking ways to preserve organized
tissues without ice crystal damage by using an entirely different
approach, namely, freezing point depression followed by extreme
supercooling [103]. He reported that by introducing 55% v/v
dimethyl sulfoxide (Me 2 SO) in a series of small steps as temperature
was commensurately reduced in a series of small steps—a technique
now referred to as “liquidus tracking” [104]—he could avoid the
toxic effects of this enormous concentration of cryoprotectant and
successfully preserve guinea pig uteri in a supercooled state at
À79
C (the temperature of subliming dry ice) with subsequent
excellent contractile function after rewarming and removal of the
Me 2 SO. This concentration of Me 2 SO is now known to be sufficient for vitrification [105], so Farrant could have actually achieved
34
Gregory M. Fahy and Brian Wowk