successful vitrification of whole organs in 1965 merely by cooling
them another 55
C or so, but at the time, this was not known, and
it was believed that cooling below À79
C would lead to ice
formation [103]. Nevertheless, Farrant achieved a new form of
“ice-free cryopreservation without vitrification,” a notable step
along the path to vitrification. The next year, similar experiments
were reported by Kemp et al. on rat, cat, and dog kidneys, which
were perfused with 55% Me 2 SO at temperatures down to À30 to
À40
C, stored at À79
C, and rewarmed by reversing the process
[106], but without tangible success.
The same year, Luyet recognized that, with respect to achieving
vitrification by rapid cooling, “we are up against a wall. Our best
chance to break through is probably to use high concentrations of
solutes” [107]. Indeed, at that time, his group, after having spent
considerable effort on characterizing the enemy that had defeated
them—ice formation—began to uncover the first evidence that
concentrated cryoprotectant solutions similar to those used by
Farrant could actually vitrify, even when cooled at slow rates
(1–3
C/min) [108, 109]. These studies continued from 1966 to
1970 [32–34, 102, 110, 111] and provided the key observations
that have enabled most modern methods of vitrification. In fact, the
first clear evidence for successful vitrification, as opposed to rapid
freezing, of living cells was published in 1968 by Rapatz and Luyet,
who showed that erythrocytes cooled at high rates in the presence
of ~5.3 M [11, 112] glycerol remained intact (did not hemolyze)
under conditions in which freeze-fracture electron microscopy
demonstrated the absence of discernible intracellular and extracellular ice crystals [11]. Before this, it is possible that some of Luyet’s
successes in recovering life after very rapid cooling and warming in
the presence of cryoprotectants (which were used to achieve dehydration prior to cooling so as to reduce the volume of water that
required vitrification but were not used to deliberately dilute the
water) [113–115] might have included some meaningful degree of
vitrification, but this is difficult to infer from available knowledge.
Ironically, the vitrification of red cells by Rapatz and Luyet, which
might have been regarded as the culmination of Luyet’s life’s work,
was not claimed as such and was noted little if at all outside of
Luyet’s laboratory for many years and is still almost never cited.
Building on the work of Farrant and on his own success with
red cells, Rapatz reported extraordinary successes with the “Farranting” of frog hearts to À79
C with 11 M ethylene glycol
(EG) in 1970 [116] and 1972 [117], obtaining good recovery
but reporting no attempt to actually vitrify these hearts and recover
them even though, unlike Farrant, he must have understood that
this might have been possible. However, he did mention from the
podium (although he did not refer to it in his published abstract
[116]) during his 1970 presentation at that year’s meeting of the
Principles of Vitrification
35
them another 55
C or so, but at the time, this was not known, and
it was believed that cooling below À79
C would lead to ice
formation [103]. Nevertheless, Farrant achieved a new form of
“ice-free cryopreservation without vitrification,” a notable step
along the path to vitrification. The next year, similar experiments
were reported by Kemp et al. on rat, cat, and dog kidneys, which
were perfused with 55% Me 2 SO at temperatures down to À30 to
À40
C, stored at À79
C, and rewarmed by reversing the process
[106], but without tangible success.
The same year, Luyet recognized that, with respect to achieving
vitrification by rapid cooling, “we are up against a wall. Our best
chance to break through is probably to use high concentrations of
solutes” [107]. Indeed, at that time, his group, after having spent
considerable effort on characterizing the enemy that had defeated
them—ice formation—began to uncover the first evidence that
concentrated cryoprotectant solutions similar to those used by
Farrant could actually vitrify, even when cooled at slow rates
(1–3
C/min) [108, 109]. These studies continued from 1966 to
1970 [32–34, 102, 110, 111] and provided the key observations
that have enabled most modern methods of vitrification. In fact, the
first clear evidence for successful vitrification, as opposed to rapid
freezing, of living cells was published in 1968 by Rapatz and Luyet,
who showed that erythrocytes cooled at high rates in the presence
of ~5.3 M [11, 112] glycerol remained intact (did not hemolyze)
under conditions in which freeze-fracture electron microscopy
demonstrated the absence of discernible intracellular and extracellular ice crystals [11]. Before this, it is possible that some of Luyet’s
successes in recovering life after very rapid cooling and warming in
the presence of cryoprotectants (which were used to achieve dehydration prior to cooling so as to reduce the volume of water that
required vitrification but were not used to deliberately dilute the
water) [113–115] might have included some meaningful degree of
vitrification, but this is difficult to infer from available knowledge.
Ironically, the vitrification of red cells by Rapatz and Luyet, which
might have been regarded as the culmination of Luyet’s life’s work,
was not claimed as such and was noted little if at all outside of
Luyet’s laboratory for many years and is still almost never cited.
Building on the work of Farrant and on his own success with
red cells, Rapatz reported extraordinary successes with the “Farranting” of frog hearts to À79
C with 11 M ethylene glycol
(EG) in 1970 [116] and 1972 [117], obtaining good recovery
but reporting no attempt to actually vitrify these hearts and recover
them even though, unlike Farrant, he must have understood that
this might have been possible. However, he did mention from the
podium (although he did not refer to it in his published abstract
[116]) during his 1970 presentation at that year’s meeting of the
Principles of Vitrification
35