glycerol + 40% bovine serum albumin at about À29
C, but did not
(and could not) measure the T G of the unfrozen solution. For
comparison, the T G of cooked meat rises from below À90
C to
À37
o C as its water content is reduced from 25.2% to 10% [35].
Another footnote to this discussion is that reference to “vitrifying sperm” in the absence of CPAs would normally imply vitrifying
not just the cells but their environment. Proponents of this
method, however, are not normally aiming to vitrify the extracellular milieu, so their use of the term “vitrify” is not synonymous with
the use of this term for other forms of biological vitrification. If
cooling were done sufficiently rapidly to vitrify the extracellular
solution, it would presumably also be rapid enough to vitrify cells
within the solution as well, but devitrification would still be
expected.
The titles of many papers cited in this section assert the achievement of vitrification in the absence of any proof. It is one thing to
postulate vitrification but something else to claim it. Luyet made
the error of claiming vitrification only to be disappointed when his
suppositions were disproven. As a matter of scientific rigor, unsubstantiated claims should in general not be made, and certainly not
in the titles of papers.
3.2 “Vitrification”
into Doubly Unstable
Glasses
and “One-Way”
Vitrification
As noted above, doubly unstable solutions are nucleated homogeneously and therefore require very high warming rates to prevent
devitrification, if devitrification can be prevented at all. Warming
vitrified samples as fast as possible, such as by immersion in a warm
bath at temperatures as high as +50
C [275] for dilute vitrification
solutions of low toxicity, may be hazardous yet still insufficient to
prevent devitrification.
However, it has been apparent for some time [226] that strict
avoidance of devitrification is not necessary [18, 89], and this has
been underscored in recent times by, for example, the successful
“vitrification” of cells using solute concentrations as low as 2 M
(15% w/v) propylene glycol plus 0.5 M (17% w/v) trehalose
[276]. This solution has a critical cooling rate on the order of
300,000
C/min, the limit of the equipment used (unstable vitrification). Considering the relationships shown in Fig. 4, the critical
warming rate must be at least 3 Â 10
8 C/min, a rate unachievable
by any currently known means of warming, and yet the cells survived. Therefore, it seems clear that the cells in these experiments
survived despite extensive devitrification.
Figure 8 documents the effect of warming rate on the survival
of cells that were rapidly cooled under conditions that led to IIF
when vitrification solutions were not used and vitrification was not
the objective. As can be seen, despite extensive IIF, cells were able
to survive in high proportions as long as they were warmed at rates
in the vicinity of 1000
C/min, which presumably “rescued” these
cells from the recrystallization of intracellular and perhaps
58
Gregory M. Fahy and Brian Wowk
C, but did not
(and could not) measure the T G of the unfrozen solution. For
comparison, the T G of cooked meat rises from below À90
C to
À37
o C as its water content is reduced from 25.2% to 10% [35].
Another footnote to this discussion is that reference to “vitrifying sperm” in the absence of CPAs would normally imply vitrifying
not just the cells but their environment. Proponents of this
method, however, are not normally aiming to vitrify the extracellular milieu, so their use of the term “vitrify” is not synonymous with
the use of this term for other forms of biological vitrification. If
cooling were done sufficiently rapidly to vitrify the extracellular
solution, it would presumably also be rapid enough to vitrify cells
within the solution as well, but devitrification would still be
expected.
The titles of many papers cited in this section assert the achievement of vitrification in the absence of any proof. It is one thing to
postulate vitrification but something else to claim it. Luyet made
the error of claiming vitrification only to be disappointed when his
suppositions were disproven. As a matter of scientific rigor, unsubstantiated claims should in general not be made, and certainly not
in the titles of papers.
3.2 “Vitrification”
into Doubly Unstable
Glasses
and “One-Way”
Vitrification
As noted above, doubly unstable solutions are nucleated homogeneously and therefore require very high warming rates to prevent
devitrification, if devitrification can be prevented at all. Warming
vitrified samples as fast as possible, such as by immersion in a warm
bath at temperatures as high as +50
C [275] for dilute vitrification
solutions of low toxicity, may be hazardous yet still insufficient to
prevent devitrification.
However, it has been apparent for some time [226] that strict
avoidance of devitrification is not necessary [18, 89], and this has
been underscored in recent times by, for example, the successful
“vitrification” of cells using solute concentrations as low as 2 M
(15% w/v) propylene glycol plus 0.5 M (17% w/v) trehalose
[276]. This solution has a critical cooling rate on the order of
300,000
C/min, the limit of the equipment used (unstable vitrification). Considering the relationships shown in Fig. 4, the critical
warming rate must be at least 3 Â 10
8 C/min, a rate unachievable
by any currently known means of warming, and yet the cells survived. Therefore, it seems clear that the cells in these experiments
survived despite extensive devitrification.
Figure 8 documents the effect of warming rate on the survival
of cells that were rapidly cooled under conditions that led to IIF
when vitrification solutions were not used and vitrification was not
the objective. As can be seen, despite extensive IIF, cells were able
to survive in high proportions as long as they were warmed at rates
in the vicinity of 1000
C/min, which presumably “rescued” these
cells from the recrystallization of intracellular and perhaps
58
Gregory M. Fahy and Brian Wowk