The possibility of intracellular vitrification given cooling rates
of up to 7.2 Â 10
5 C/min [271] seems plausible, but has to be
compared to the fact that the estimated cooling rate required to
vitrify 0.3 M glycerol in an isotonic salt solution, which would have
a lower activity of water than isotonic cells including sperm cells,
can be estimated from curves originating from the work of Toner to
be on the order of 2 Â 10
6 to 2 Â 10
7 C/min or more [18]. In
addition, viability was found to be more or less independent of
cooling rate, which is consistent with the survival of IIF. A general
problem with attempting to prove that cells can survive vitrification
without cryoprotectants is that if cooling is sufficiently rapid, IIF
leads to ice crystals that are too small to kill the cell outright, and if
warming is sufficiently rapid to preclude recrystallization, these
crystals will continue to be innocuous during rewarming, so IIF
can be mistaken for vitrification if the main criterion for vitrification
is survival [18, 226] (see further discussion and examples in the
next section). Although the water content of sperm is relatively low,
the extent to which that water is immobilized by contact with
intracellular solutes and structures may not be reflected by the
water content per se.
Morris [274] checked human sperm for intracellular ice using
freeze-fracture freeze-etch electron microscopy and freeze substitution after freezing at various rates after previously equilibrating
them with ice at À7
C with and without glycerol pretreatment. He
observed no clear evidence for intracellular ice, even after rewarming to À40
C. The results, though, do not speak directly to the
question of whether sperm can be vitrified without pCPAs, because
cells pre-equilibrated at À7
C were also equilibrated, prior to rapid
cooling, with either about 20% glycerol, both extracellularly and
intracellularly, or with an osmoticum about 13 times more concentrated (~3.8 Osm) than native sperm contents, which would tend
to withdraw about 92% of the freezable water from the sperm.
Therefore, both pre-exposures would dramatically increase resistance to IIF compared to sperm quenched under isotonic conditions [268]. Moreover, sperm frozen to À7
C and below without
glycerol were not viable. In this regard, sperm are no different than
muscle: slow freezing of muscle results in vitrification of the
unfrozen residual liquid once the water content has been reduced
to about 20% by mass [35], but muscle cells rendered vitrifiable by
such extreme dehydration would not be viable.
Morris has estimated intracellular T h values for isotonic, pCPAfree sperm as a function of intracellular protein concentration,
finding T h ~ À43
C for 30% protein and T h ~ À58
C for 60%
protein [268]. However, a T h of À58
C would be the equivalent of
~30% w/w glycerol, which would require a T m of ~À7
C, versus
the À0.5
C characteristic of isotonic cells, and Morris estimates the
actual intracellular protein concentration in cells as around 20%. He
measured the T G of a previously freeze-concentrated solution of 10%
Principles of Vitrification
57
of up to 7.2 Â 10
5 C/min [271] seems plausible, but has to be
compared to the fact that the estimated cooling rate required to
vitrify 0.3 M glycerol in an isotonic salt solution, which would have
a lower activity of water than isotonic cells including sperm cells,
can be estimated from curves originating from the work of Toner to
be on the order of 2 Â 10
6 to 2 Â 10
7 C/min or more [18]. In
addition, viability was found to be more or less independent of
cooling rate, which is consistent with the survival of IIF. A general
problem with attempting to prove that cells can survive vitrification
without cryoprotectants is that if cooling is sufficiently rapid, IIF
leads to ice crystals that are too small to kill the cell outright, and if
warming is sufficiently rapid to preclude recrystallization, these
crystals will continue to be innocuous during rewarming, so IIF
can be mistaken for vitrification if the main criterion for vitrification
is survival [18, 226] (see further discussion and examples in the
next section). Although the water content of sperm is relatively low,
the extent to which that water is immobilized by contact with
intracellular solutes and structures may not be reflected by the
water content per se.
Morris [274] checked human sperm for intracellular ice using
freeze-fracture freeze-etch electron microscopy and freeze substitution after freezing at various rates after previously equilibrating
them with ice at À7
C with and without glycerol pretreatment. He
observed no clear evidence for intracellular ice, even after rewarming to À40
C. The results, though, do not speak directly to the
question of whether sperm can be vitrified without pCPAs, because
cells pre-equilibrated at À7
C were also equilibrated, prior to rapid
cooling, with either about 20% glycerol, both extracellularly and
intracellularly, or with an osmoticum about 13 times more concentrated (~3.8 Osm) than native sperm contents, which would tend
to withdraw about 92% of the freezable water from the sperm.
Therefore, both pre-exposures would dramatically increase resistance to IIF compared to sperm quenched under isotonic conditions [268]. Moreover, sperm frozen to À7
C and below without
glycerol were not viable. In this regard, sperm are no different than
muscle: slow freezing of muscle results in vitrification of the
unfrozen residual liquid once the water content has been reduced
to about 20% by mass [35], but muscle cells rendered vitrifiable by
such extreme dehydration would not be viable.
Morris has estimated intracellular T h values for isotonic, pCPAfree sperm as a function of intracellular protein concentration,
finding T h ~ À43
C for 30% protein and T h ~ À58
C for 60%
protein [268]. However, a T h of À58
C would be the equivalent of
~30% w/w glycerol, which would require a T m of ~À7
C, versus
the À0.5
C characteristic of isotonic cells, and Morris estimates the
actual intracellular protein concentration in cells as around 20%. He
measured the T G of a previously freeze-concentrated solution of 10%
Principles of Vitrification
57