the living system or its environment does not remain amorphous
during warming [89].
Describing rapid freezing or unstable vitrification methods
simply as “vitrification” is not sufficiently accurate, obscures the
true physical nature of the process being employed, and should be
avoided.
Recently, ultrarapid warming techniques for small systems
based on laser warming [280] have opened up new possibilities
for avoiding devitrification-related injury, particularly when combined with cellular osmotic dehydration prior to cooling
[281]. When combined with gold nanorod microinjection, laser
warming was able to achieve a warming rate of 1.4 Â 10
7 C/min
even in relatively massive but transparent zebrafish embryos,
achieving improved viability [282].
3.3 Carrier Solutions
and Cryoprotectants
All cells normally survive in an environment to which they have
been adapted. In the mammalian body, this environment has some
common characteristics, including, typically, a total osmolality a
little less than 300 mOsm (which depresses the freezing point to
about À0.55
C), a high sodium concentration (~145 mM), a low
potassium concentration (~4.5 mM), a high chloride content, a pH
in the vicinity of 7.4, and a variety of other electrolytes, proteins,
signaling molecules, etc. To survive exposure to low-temperature
conditions, this environment must in some form be maintained
within viable limits, and that is the function of the “carrier solution,” which is the physiological support medium in which cryoprotectants are dissolved. Cells suspended in pCPAs alone would
not be able to maintain their volume, pH, ionic content, and
membrane integrity, so there must be a basic solution that is maintained around the cells in both the presence and absence of cryoprotectants. The carrier solution in a sense carries the
cryoprotectants to and from living cells while allowing them to
avoid injury that is unrelated to the cryoprotectants per
se. Carrier solution compositions vary widely, but all are designed
with this basic supportive role in mind. Cryoprotectants must
generally be combined with carrier solutions to enable vitrification.
The toxic effects of cryoprotectants have been shown to
depend on the choice of the carrier solution [178, 283–
285]. Often the reason for this dependence is unclear, but carriers
that do not support cell viability under hypothermic conditions
may add extraneous hypothermic injury to any injury that may be
due to CPA exposure per se.
As noted above (Subheading 2.5), the carrier solution also
plays an important role in limiting ice nucleation and ice growth
rates during both cooling and warming. An influence of this effect
on survival rates, though, remains to be demonstrated.
60
Gregory M. Fahy and Brian Wowk
during warming [89].
Describing rapid freezing or unstable vitrification methods
simply as “vitrification” is not sufficiently accurate, obscures the
true physical nature of the process being employed, and should be
avoided.
Recently, ultrarapid warming techniques for small systems
based on laser warming [280] have opened up new possibilities
for avoiding devitrification-related injury, particularly when combined with cellular osmotic dehydration prior to cooling
[281]. When combined with gold nanorod microinjection, laser
warming was able to achieve a warming rate of 1.4 Â 10
7 C/min
even in relatively massive but transparent zebrafish embryos,
achieving improved viability [282].
3.3 Carrier Solutions
and Cryoprotectants
All cells normally survive in an environment to which they have
been adapted. In the mammalian body, this environment has some
common characteristics, including, typically, a total osmolality a
little less than 300 mOsm (which depresses the freezing point to
about À0.55
C), a high sodium concentration (~145 mM), a low
potassium concentration (~4.5 mM), a high chloride content, a pH
in the vicinity of 7.4, and a variety of other electrolytes, proteins,
signaling molecules, etc. To survive exposure to low-temperature
conditions, this environment must in some form be maintained
within viable limits, and that is the function of the “carrier solution,” which is the physiological support medium in which cryoprotectants are dissolved. Cells suspended in pCPAs alone would
not be able to maintain their volume, pH, ionic content, and
membrane integrity, so there must be a basic solution that is maintained around the cells in both the presence and absence of cryoprotectants. The carrier solution in a sense carries the
cryoprotectants to and from living cells while allowing them to
avoid injury that is unrelated to the cryoprotectants per
se. Carrier solution compositions vary widely, but all are designed
with this basic supportive role in mind. Cryoprotectants must
generally be combined with carrier solutions to enable vitrification.
The toxic effects of cryoprotectants have been shown to
depend on the choice of the carrier solution [178, 283–
285]. Often the reason for this dependence is unclear, but carriers
that do not support cell viability under hypothermic conditions
may add extraneous hypothermic injury to any injury that may be
due to CPA exposure per se.
As noted above (Subheading 2.5), the carrier solution also
plays an important role in limiting ice nucleation and ice growth
rates during both cooling and warming. An influence of this effect
on survival rates, though, remains to be demonstrated.
60
Gregory M. Fahy and Brian Wowk