whereas accelerating exposure of the renal cortex to the CPA and
then holding the cortex at the highest concentrations long enough
for the medulla to “catch up” can be lethal [22, 236]. As a general
rule, the osmotic protocol for each system should be tailored to the
specific needs and characteristics of that system.
3.5 Procedures
for Avoiding
Cryoprotectant
Toxicity
The central problem of vitrification has always been inducing living
cells to tolerate enormous concentrations of CPAs and low concentrations of water. In 1984, Fahy et al. suggested seven
approaches to controlling cryoprotectant toxicity in vitrification
procedures, i.e.:
Avoid osmotic injury.
Employ cryoprotectant mixtures so their mutual dilution minimizes specific sources of toxicity.
Use one or more npCPAs to allow reduction in the intracellular
pCPA concentration (see Subheading 3.6 for more details).
Maintain temperature as low as possible.
Select an appropriate carrier solution.
Keep exposure time to the CPA to a minimum.
Employ cryoprotectant toxicity neutralization, when possible
(see Subheading 3.7).
Implicit in this list at the time was also the very first step in
designing a vitrification solution, and that is to determine exactly
how much CPA is needed (C V ). Extensive lists of solutions that are,
within 1% w/v total concentration, exactly sufficient in concentration to avoid visible ice crystals (i.e., that are at their C V ) on cooling
at about 10
C/min have been published (e.g., [87, 105, 166]),
and means of interpolating between known solutions to estimate
the C V s of arbitrary mixtures of CPAs have also been described
(e.g., [179, 227]). However, some investigators determine C V only
to the nearest 5%, potentially exposing their cells to as much as 4%
w/v or v/v (or w/w) more CPA than needed, which may significantly [295] and unnecessarily increase the total toxicity observed.
C V is a function of solution composition, cooling rate, and applied
pressure [87, 129, 131, 166, 233, 296], so it must frequently be
redetermined for new circumstances.
These methods have been used successfully, but since 1984,
seven additional approaches have been added or at least proposed:
use ice blockers to reduce the overall quantity of pCPA required
[50, 51]; use methoxylated CPAs in moderation [297]; employ
creative addition and washout methods to minimize the overall
“cost function” of toxicity for the solution in question when the
system is a cell suspension or simple tissue [298–300]; preferentially
employ weak glass formers [245]; augment the heat shock response
[301]; block biochemical pathways that contribute to cryoprotectant toxicity [302]; and, for whole organ cryoprotection, use special perfusion techniques to speed equilibration [236, 303] and
reduce injury [303, 304]. Organ perfusion techniques are still
66
Gregory M. Fahy and Brian Wowk
then holding the cortex at the highest concentrations long enough
for the medulla to “catch up” can be lethal [22, 236]. As a general
rule, the osmotic protocol for each system should be tailored to the
specific needs and characteristics of that system.
3.5 Procedures
for Avoiding
Cryoprotectant
Toxicity
The central problem of vitrification has always been inducing living
cells to tolerate enormous concentrations of CPAs and low concentrations of water. In 1984, Fahy et al. suggested seven
approaches to controlling cryoprotectant toxicity in vitrification
procedures, i.e.:
Avoid osmotic injury.
Employ cryoprotectant mixtures so their mutual dilution minimizes specific sources of toxicity.
Use one or more npCPAs to allow reduction in the intracellular
pCPA concentration (see Subheading 3.6 for more details).
Maintain temperature as low as possible.
Select an appropriate carrier solution.
Keep exposure time to the CPA to a minimum.
Employ cryoprotectant toxicity neutralization, when possible
(see Subheading 3.7).
Implicit in this list at the time was also the very first step in
designing a vitrification solution, and that is to determine exactly
how much CPA is needed (C V ). Extensive lists of solutions that are,
within 1% w/v total concentration, exactly sufficient in concentration to avoid visible ice crystals (i.e., that are at their C V ) on cooling
at about 10
C/min have been published (e.g., [87, 105, 166]),
and means of interpolating between known solutions to estimate
the C V s of arbitrary mixtures of CPAs have also been described
(e.g., [179, 227]). However, some investigators determine C V only
to the nearest 5%, potentially exposing their cells to as much as 4%
w/v or v/v (or w/w) more CPA than needed, which may significantly [295] and unnecessarily increase the total toxicity observed.
C V is a function of solution composition, cooling rate, and applied
pressure [87, 129, 131, 166, 233, 296], so it must frequently be
redetermined for new circumstances.
These methods have been used successfully, but since 1984,
seven additional approaches have been added or at least proposed:
use ice blockers to reduce the overall quantity of pCPA required
[50, 51]; use methoxylated CPAs in moderation [297]; employ
creative addition and washout methods to minimize the overall
“cost function” of toxicity for the solution in question when the
system is a cell suspension or simple tissue [298–300]; preferentially
employ weak glass formers [245]; augment the heat shock response
[301]; block biochemical pathways that contribute to cryoprotectant toxicity [302]; and, for whole organ cryoprotection, use special perfusion techniques to speed equilibration [236, 303] and
reduce injury [303, 304]. Organ perfusion techniques are still
66
Gregory M. Fahy and Brian Wowk