vitrified systems. The former may be more susceptible to mechanical or chemical sources of injury, the nature and meaning of the
glassy state may be different in the dried and hydrated states, and
deterioration may be measured at much higher absolute temperatures in dried systems than in hydrated vitrified systems. Free
radicals may be more diffusible in systems that include a great
deal of empty space, and at higher temperatures, damaging membrane phase transitions can occur at temperatures well below the
nominal global T G of the system [385]. According to Sun, however, deterioration of dried liposomes can sometimes be stopped by
using more sugar prior to drying, perhaps because the extra sugar
provides space between the liposomes so that they don’t fuse
[383]. This may be more analogous to a hydrated cryoprotected
system, in which water, the smallest major biological molecule, and
pCPA fill in more gaps and bond the system together more strongly
and in which pCPAs may serve as targets for free radicals, reducing
their interactions with biological targets [387]. For the time being,
it is not clear that hydrated cryoprotected systems deteriorate
appreciably over timescales of even hundreds of years at À196
C.
Studies of the effects of irradiation of frozen cells compared against
normal levels of background radiation have led to the prediction
that it would require over 32,000 years of background radiation to
kill 90% of CHO cells frozen in 10% Me 2 SO [387] and more than
500 years to have even a slight effect on viability of frozen mouse
embryos [388]. No similar studies of irradiated vitrified systems,
which may be more protected from background radiation, have
apparently been carried out.
3.11 Proteins at Low
Temperatures
We conclude by noting a little-referenced area of molecular biology
that has a direct bearing on the principles of biological vitrification
and that may one day provide additional insights that will help to
guide the cryobiologist to more successful vitrification methods.
This field of research, called “cryoenzymology,” has provided a
significant body of literature documenting the ability of mixed
cosolvent systems comprising water and molecules that depress its
freezing point to maintain protein secondary, tertiary, and quaternary structure sufficiently well in some cases to enable normal
enzymatic catalysis to proceed, albeit at greatly reduced reaction
rates, at temperatures as low as À70
C [389, 390]. Because vitrification preserves cells and molecules in the absence of ice, cryoenzymology offers many direct windows on phenomena that may
affect the success of vitrification, including changing dielectric
constants and pK a values with decreasing temperature and increasing cryoprotectant concentrations [390], which may bear on such
phenomena as cold denaturation and chilling injury during vitrification. On the other hand, one of the observations made is that
although protein cold denaturation is a real phenomenon [391–
393], it can be prevented in some cases by rapid cooling to temperatures too low to favor it kinetically [390, 391], and
Principles of Vitrification
81
glassy state may be different in the dried and hydrated states, and
deterioration may be measured at much higher absolute temperatures in dried systems than in hydrated vitrified systems. Free
radicals may be more diffusible in systems that include a great
deal of empty space, and at higher temperatures, damaging membrane phase transitions can occur at temperatures well below the
nominal global T G of the system [385]. According to Sun, however, deterioration of dried liposomes can sometimes be stopped by
using more sugar prior to drying, perhaps because the extra sugar
provides space between the liposomes so that they don’t fuse
[383]. This may be more analogous to a hydrated cryoprotected
system, in which water, the smallest major biological molecule, and
pCPA fill in more gaps and bond the system together more strongly
and in which pCPAs may serve as targets for free radicals, reducing
their interactions with biological targets [387]. For the time being,
it is not clear that hydrated cryoprotected systems deteriorate
appreciably over timescales of even hundreds of years at À196
C.
Studies of the effects of irradiation of frozen cells compared against
normal levels of background radiation have led to the prediction
that it would require over 32,000 years of background radiation to
kill 90% of CHO cells frozen in 10% Me 2 SO [387] and more than
500 years to have even a slight effect on viability of frozen mouse
embryos [388]. No similar studies of irradiated vitrified systems,
which may be more protected from background radiation, have
apparently been carried out.
3.11 Proteins at Low
Temperatures
We conclude by noting a little-referenced area of molecular biology
that has a direct bearing on the principles of biological vitrification
and that may one day provide additional insights that will help to
guide the cryobiologist to more successful vitrification methods.
This field of research, called “cryoenzymology,” has provided a
significant body of literature documenting the ability of mixed
cosolvent systems comprising water and molecules that depress its
freezing point to maintain protein secondary, tertiary, and quaternary structure sufficiently well in some cases to enable normal
enzymatic catalysis to proceed, albeit at greatly reduced reaction
rates, at temperatures as low as À70
C [389, 390]. Because vitrification preserves cells and molecules in the absence of ice, cryoenzymology offers many direct windows on phenomena that may
affect the success of vitrification, including changing dielectric
constants and pK a values with decreasing temperature and increasing cryoprotectant concentrations [390], which may bear on such
phenomena as cold denaturation and chilling injury during vitrification. On the other hand, one of the observations made is that
although protein cold denaturation is a real phenomenon [391–
393], it can be prevented in some cases by rapid cooling to temperatures too low to favor it kinetically [390, 391], and
Principles of Vitrification
81