exposure at 37
C better than wild-type cells. Because the sites of
transposon insertion into the genome can be determined by
sequencing, they were able to identify genes containing or flanking
the insertion sites in each case. Ten genes were thereby identified
with apparent roles in the modulation of cryoprotectant toxicity, of
which two are related to MYC signaling, which had previously been
found to change in Guan’s microarray study [61] and is a regulator
of ribosome biogenesis and protein synthesis, and two others may
activate and stabilize MYC. Two of the other mutated genes, Hsbp1
and Ywhag, normally restrain stress responses. The relevance of
screening at 37
C was in this case validated by showing that
M22-resistant mutants were also resistant to Me 2 SO exposure
and, most importantly, survived in significantly higher numbers
when frozen and thawed with Me 2 SO than did non-mutated
cells. Intriguingly, mutated ESCs can often be grown into adults,
whose organs might in this case be more resistant to cryoprotectants than ordinary organs.
A similar model has also been applied to the mitigation of M22
toxicity in whole C. elegans [348]. Two mutants, involving age-1
and daf-2, which were formerly found to extend lifespan in this
species and to activate stress responses, were found to be more
resistant to M22 than wild-type worms, with high statistical significance. In addition, a drug, afatinib, which may mimic some of the
effects of toxicity-blocking mutations, inhibited the toxicity of 10%
M22 in C. elegans at a drug concentration of 100 nM with high
statistical significance.
The mutation screening approach has the advantage of identifying exact genes that are directly related to toxicity, whereas microarray studies identify large numbers of candidate genes that may or
may not be primary and whose modulation may be either compensatory or contributory to the observed injury. Identification of
specific relevant genes then enables a search for drugs that will
induce the same biochemical effects as the mutations. This
approach therefore opens up remarkable new possibilities for both
understanding and correcting cryoprotectant toxicity.
In summary, although our understanding of cryoprotectant
toxicity remains in its infancy, the power of molecular biology to
elucidate and interrupt mechanisms of cryoprotectant toxicity,
though long deferred, is finally beginning to be exercised. The
possibilities are potentially transformative.
3.9 Chilling Injury
and Its Modification or
Avoidance
Chilling injury is observed both in nature at temperatures above
0
C [349–351] and in the laboratory at temperatures well below
zero [23, 61, 64, 65]. It has been linked to phase changes in
membranes [82, 83, 352–355] and associated defects in membrane
permeability [354, 356, 357] and can be blocked in some cases by
directly modifying cell membrane composition [83, 358, 359], by
using antifreeze proteins [356, 360], or by using genetic
76
Gregory M. Fahy and Brian Wowk
C better than wild-type cells. Because the sites of
transposon insertion into the genome can be determined by
sequencing, they were able to identify genes containing or flanking
the insertion sites in each case. Ten genes were thereby identified
with apparent roles in the modulation of cryoprotectant toxicity, of
which two are related to MYC signaling, which had previously been
found to change in Guan’s microarray study [61] and is a regulator
of ribosome biogenesis and protein synthesis, and two others may
activate and stabilize MYC. Two of the other mutated genes, Hsbp1
and Ywhag, normally restrain stress responses. The relevance of
screening at 37
C was in this case validated by showing that
M22-resistant mutants were also resistant to Me 2 SO exposure
and, most importantly, survived in significantly higher numbers
when frozen and thawed with Me 2 SO than did non-mutated
cells. Intriguingly, mutated ESCs can often be grown into adults,
whose organs might in this case be more resistant to cryoprotectants than ordinary organs.
A similar model has also been applied to the mitigation of M22
toxicity in whole C. elegans [348]. Two mutants, involving age-1
and daf-2, which were formerly found to extend lifespan in this
species and to activate stress responses, were found to be more
resistant to M22 than wild-type worms, with high statistical significance. In addition, a drug, afatinib, which may mimic some of the
effects of toxicity-blocking mutations, inhibited the toxicity of 10%
M22 in C. elegans at a drug concentration of 100 nM with high
statistical significance.
The mutation screening approach has the advantage of identifying exact genes that are directly related to toxicity, whereas microarray studies identify large numbers of candidate genes that may or
may not be primary and whose modulation may be either compensatory or contributory to the observed injury. Identification of
specific relevant genes then enables a search for drugs that will
induce the same biochemical effects as the mutations. This
approach therefore opens up remarkable new possibilities for both
understanding and correcting cryoprotectant toxicity.
In summary, although our understanding of cryoprotectant
toxicity remains in its infancy, the power of molecular biology to
elucidate and interrupt mechanisms of cryoprotectant toxicity,
though long deferred, is finally beginning to be exercised. The
possibilities are potentially transformative.
3.9 Chilling Injury
and Its Modification or
Avoidance
Chilling injury is observed both in nature at temperatures above
0
C [349–351] and in the laboratory at temperatures well below
zero [23, 61, 64, 65]. It has been linked to phase changes in
membranes [82, 83, 352–355] and associated defects in membrane
permeability [354, 356, 357] and can be blocked in some cases by
directly modifying cell membrane composition [83, 358, 359], by
using antifreeze proteins [356, 360], or by using genetic
76
Gregory M. Fahy and Brian Wowk