engineering [361, 362] to inhibit these phase transitions and associated membrane leakage. Chilling injury may also result from
protein denaturation based on protection by prior heat shock
[363], the production of heat shock proteins in response to chilling
[363], and the production of “cold shock proteins” in response to
chilling as well [364, 365]. Also suggestive is the observation that
the unfolded protein response (ER stress response) is induced by
18 h of cold storage of human corneal endothelial cells and inhibited using the specific blocking agent, salubrinal [366]. However,
chilling injury is generally observed to be an immediate or shortterm response rather than a delayed response to temperature reduction [23, 64, 65].
None of the above mechanistic and interventional studies
involved systems that were saturated with a multi-molar concentration of CPA(s), and chilling injury associated with vitrification is
typically observed primarily or exclusively at temperatures far below
those of the lipid phase transition temperatures and denaturation
phenomena noted above [23, 61, 64, 65, 145]. Further, one study
showed that chilling injury (though not thermal shock) could be
prevented in polymorphonuclear cells by Me 2 SO [367].
Nevertheless, one DNA microarray study of chilling injury in
the context of vitrification has been done, and it verified changes
suggestive of the ER stress/unfolded protein response and altered
lipid metabolism [61]. Precision-cut rat liver slices loaded with
either of two vitrification solutions showed no drop in ATP content
compared to controls when held at 0
C, but a 20–30% drop after
cooling without freezing to À15
C for 10 min (as detected after
incubation at 37
C following CPA washout). Principal component
analysis indicated clear separation between the effects of CPA
administration and the effects of chilling in the presence of CPA.
Comparing CPA treatment to CPA treatment plus chilling, 1108
transcripts changed in abundance with chilling, but of these, only
31 increased more than 1.5-fold and only 6 decreased more than
1.5-fold, so the changes observed were in general mild, in keeping
with the mild change in ATP content, and likely were indicative of
the first changes induced by chilling. The primary observations
were an increase in heat shock protein and heat shock factor transcripts, an increase in ribosomal RNA transcripts (which would
favor more protein synthesis to replace denatured proteins), a lack
of activation of apoptotic pathways (suggesting ER stress did not
reach levels sufficient to induce apoptosis), activation of DNA
damage-sensing genes, activation of two of the three MAP kinase
stress pathways (involving increased JNK and ERK signaling without increased p38 signaling), changes that tend to reduce cholesterol synthesis and remove cholesterol from the cell membrane and
transfer it to the endoplasmic reticulum, and, rather paradoxically,
Principles of Vitrification
77
protein denaturation based on protection by prior heat shock
[363], the production of heat shock proteins in response to chilling
[363], and the production of “cold shock proteins” in response to
chilling as well [364, 365]. Also suggestive is the observation that
the unfolded protein response (ER stress response) is induced by
18 h of cold storage of human corneal endothelial cells and inhibited using the specific blocking agent, salubrinal [366]. However,
chilling injury is generally observed to be an immediate or shortterm response rather than a delayed response to temperature reduction [23, 64, 65].
None of the above mechanistic and interventional studies
involved systems that were saturated with a multi-molar concentration of CPA(s), and chilling injury associated with vitrification is
typically observed primarily or exclusively at temperatures far below
those of the lipid phase transition temperatures and denaturation
phenomena noted above [23, 61, 64, 65, 145]. Further, one study
showed that chilling injury (though not thermal shock) could be
prevented in polymorphonuclear cells by Me 2 SO [367].
Nevertheless, one DNA microarray study of chilling injury in
the context of vitrification has been done, and it verified changes
suggestive of the ER stress/unfolded protein response and altered
lipid metabolism [61]. Precision-cut rat liver slices loaded with
either of two vitrification solutions showed no drop in ATP content
compared to controls when held at 0
C, but a 20–30% drop after
cooling without freezing to À15
C for 10 min (as detected after
incubation at 37
C following CPA washout). Principal component
analysis indicated clear separation between the effects of CPA
administration and the effects of chilling in the presence of CPA.
Comparing CPA treatment to CPA treatment plus chilling, 1108
transcripts changed in abundance with chilling, but of these, only
31 increased more than 1.5-fold and only 6 decreased more than
1.5-fold, so the changes observed were in general mild, in keeping
with the mild change in ATP content, and likely were indicative of
the first changes induced by chilling. The primary observations
were an increase in heat shock protein and heat shock factor transcripts, an increase in ribosomal RNA transcripts (which would
favor more protein synthesis to replace denatured proteins), a lack
of activation of apoptotic pathways (suggesting ER stress did not
reach levels sufficient to induce apoptosis), activation of DNA
damage-sensing genes, activation of two of the three MAP kinase
stress pathways (involving increased JNK and ERK signaling without increased p38 signaling), changes that tend to reduce cholesterol synthesis and remove cholesterol from the cell membrane and
transfer it to the endoplasmic reticulum, and, rather paradoxically,
Principles of Vitrification
77