vitrifying cells and recovering them in a viable state upon rewarming. The problem of vitrifying cells without pCPAs remains a topic
of some current interest and discussion, and a brief examination of
this question is useful for illustrating additional principles relevant
to cryopreservation by vitrification.
The vitrification tendency of cryoprotectant solutions has been
studied extensively as a function of cryoprotectant concentration,
but living cells contain solutes in addition to any cryoprotectants
they may take up, and these natural solutes definitely augment the
vitrification tendency of cytoplasm in the presence of added pCPAs
[11, 105, 179, 262]. The quantitative contribution of intracellular
solutes to intracellular vitrification in different cells or in different
organelles has, however, not been well studied. Cytoplasm typically
contains 15% w/w protein and just 80% water, the endoplasmic
reticulum contains only 65% water, nuclei contain only 61% water,
and mitochondria contain only 59% water [226]. Exactly how these
low water contents influence vitrification tendency deserves to be
considered more carefully. However, since most cells and their
organelles are in osmotic equilibrium at ~300 milliosmolal
(mOsm), they do not have an extraordinary water activity and
readily experience IIF and behave very much as though their
water is present as an ideal dilute solution [157–159, 263].
Ice formation is also affected by the presence of solid surfaces,
whose organization of local water structure tends to inhibit ice
formation. According to Meryman’s discussion of the work of
Hori (1956), the spontaneous freezing temperature of water
between glass plates separated by 10 μm is À30
C, and a plate
separation on the order of 0.14 μm results in no ice formation even
at À100
C and a negligible water vapor pressure [264]. Water
structuring by other types of surface has been investigated at some
length [265, 266]. The water in intracellular compartments may be
influenced by this same general type of effect near the plasma
membrane, organelle membranes, and the cytoskeleton, further
favoring vitrification, and the effect might be particularly significant
for sperm considering their highly elongated, thin shape and
packed DNA.
The water content of sperm, as estimated from their osmotically inactive volume (the b value in the Boyle-van ’t Hoff equation
described below), is just 23–55% according to Isachenko et al.
[267] or 60% according to Morris [268]. This naturally low water
content has led to speculation that sperm might be vitrifiable
internally in the absence of pCPAs even at relatively low cooling
rates (~150–250
C/min [267]), but direct evidence for this speculation is lacking. Others have cooled sperm at high rates without
pCPAs and obtained survival, but again have not verified that the
survival was due to intracellular vitrification and the prevention of
devitrification [269–272], and another report notes ice formation
in the sperm tail, midpiece, and neck after rapid freezing [273].
56
Gregory M. Fahy and Brian Wowk
of some current interest and discussion, and a brief examination of
this question is useful for illustrating additional principles relevant
to cryopreservation by vitrification.
The vitrification tendency of cryoprotectant solutions has been
studied extensively as a function of cryoprotectant concentration,
but living cells contain solutes in addition to any cryoprotectants
they may take up, and these natural solutes definitely augment the
vitrification tendency of cytoplasm in the presence of added pCPAs
[11, 105, 179, 262]. The quantitative contribution of intracellular
solutes to intracellular vitrification in different cells or in different
organelles has, however, not been well studied. Cytoplasm typically
contains 15% w/w protein and just 80% water, the endoplasmic
reticulum contains only 65% water, nuclei contain only 61% water,
and mitochondria contain only 59% water [226]. Exactly how these
low water contents influence vitrification tendency deserves to be
considered more carefully. However, since most cells and their
organelles are in osmotic equilibrium at ~300 milliosmolal
(mOsm), they do not have an extraordinary water activity and
readily experience IIF and behave very much as though their
water is present as an ideal dilute solution [157–159, 263].
Ice formation is also affected by the presence of solid surfaces,
whose organization of local water structure tends to inhibit ice
formation. According to Meryman’s discussion of the work of
Hori (1956), the spontaneous freezing temperature of water
between glass plates separated by 10 μm is À30
C, and a plate
separation on the order of 0.14 μm results in no ice formation even
at À100
C and a negligible water vapor pressure [264]. Water
structuring by other types of surface has been investigated at some
length [265, 266]. The water in intracellular compartments may be
influenced by this same general type of effect near the plasma
membrane, organelle membranes, and the cytoskeleton, further
favoring vitrification, and the effect might be particularly significant
for sperm considering their highly elongated, thin shape and
packed DNA.
The water content of sperm, as estimated from their osmotically inactive volume (the b value in the Boyle-van ’t Hoff equation
described below), is just 23–55% according to Isachenko et al.
[267] or 60% according to Morris [268]. This naturally low water
content has led to speculation that sperm might be vitrifiable
internally in the absence of pCPAs even at relatively low cooling
rates (~150–250
C/min [267]), but direct evidence for this speculation is lacking. Others have cooled sperm at high rates without
pCPAs and obtained survival, but again have not verified that the
survival was due to intracellular vitrification and the prevention of
devitrification [269–272], and another report notes ice formation
in the sperm tail, midpiece, and neck after rapid freezing [273].
56
Gregory M. Fahy and Brian Wowk