3.10 Storage
in the Vitreous or
Near-Vitreous State
Little is known about the safety of various durations of storage in
the vitreous state at temperatures in the vicinity of T G , but this is an
important topic for several reasons. First, the risk of fracture formation increases as vitreous samples are cooled to the temperature of
liquid nitrogen, and fractures may damage organs, tissues, oocytes,
embryos, and other systems as well as create sites of ice nucleation
[182] that may indirectly damage vitrified cells during warming.
Second, liquid nitrogen immersion has a number of practical,
safety, and potential contamination issues that could be avoided
by storing in the vapor phase if this were known to be safe. Therefore, one would like to know how far below T G a sample must be
cooled to protect it for long-term storage and to verify that this
temperature is still warm enough to minimize the risk of fracturing.
Empirically, Rowe found no difference in stability of rapidly
frozen red cells between À165
C and À196
C over 16 years
[379]. Valeri and Pivacek found no difference in the recovery of
frozen peripheral blood mononuclear cells stored at À135, À150,
and À196
C for 2–2.4 years [380]. Red cells frozen in 40–45%
w/v glycerol were stored successfully for 21 years even at À80
C
[381]. Most pertinently, Song et al. [382] reported that vitrified
rabbit jugular veins (T G ~ À123
C) stored at À130
C for 4 weeks
or for 4 months or stored below À160
C in liquid nitrogen vapor
either for 4 weeks or for 4 months all recovered as well as veins
stored for only 24 h and approached the functionality of fresh
controls. Heart valves and cartilage yielded similar results
[382]. Our laboratory has stored rabbit hippocampal slices under
isothermal conditions in the vicinity of À145
C (T G ~ À124
C)
for months as well, without detectable deterioration [248]. In Song
et al.’s experience, there was no visual development of ice during
storage, and freeze substitution showed no ice development after
5 months of storage in liquid nitrogen vapor [382]. Although
indirect, these observations are consistent with the possibility of
storing vitrified systems not far below T G .
Fahy and Rall [18] proposed that the kinetics of storage injury
at different temperatures might be predictable if biological deterioration is rate-limited by molecular motions and therefore by
increasing viscosity as temperature falls. Based on this assumption
and the relationship between T and viscosity in vitrifiable solutions,
the time t required for a given amount of deterioration to be
observed at storage temperature T can be related to the time t 1
required for the same amount of deterioration to be observed at a
higher reference temperature T 1 by the following equation:
t ¼ t 1 T 1 =T
ð
Þ exp B 1= T À T 0
ð
Þ
ð
ÞÀ1= T 1 À T 0
ð
Þ
½
½
,
where T 0 and B are empirical constants. The time required for
deterioration is not adjusted in this equation for the time required
for cooling to the storage temperature, but deterioration accumulated as a function of temperature during cooling at 1
C/min has
Principles of Vitrification
79
in the Vitreous or
Near-Vitreous State
Little is known about the safety of various durations of storage in
the vitreous state at temperatures in the vicinity of T G , but this is an
important topic for several reasons. First, the risk of fracture formation increases as vitreous samples are cooled to the temperature of
liquid nitrogen, and fractures may damage organs, tissues, oocytes,
embryos, and other systems as well as create sites of ice nucleation
[182] that may indirectly damage vitrified cells during warming.
Second, liquid nitrogen immersion has a number of practical,
safety, and potential contamination issues that could be avoided
by storing in the vapor phase if this were known to be safe. Therefore, one would like to know how far below T G a sample must be
cooled to protect it for long-term storage and to verify that this
temperature is still warm enough to minimize the risk of fracturing.
Empirically, Rowe found no difference in stability of rapidly
frozen red cells between À165
C and À196
C over 16 years
[379]. Valeri and Pivacek found no difference in the recovery of
frozen peripheral blood mononuclear cells stored at À135, À150,
and À196
C for 2–2.4 years [380]. Red cells frozen in 40–45%
w/v glycerol were stored successfully for 21 years even at À80
C
[381]. Most pertinently, Song et al. [382] reported that vitrified
rabbit jugular veins (T G ~ À123
C) stored at À130
C for 4 weeks
or for 4 months or stored below À160
C in liquid nitrogen vapor
either for 4 weeks or for 4 months all recovered as well as veins
stored for only 24 h and approached the functionality of fresh
controls. Heart valves and cartilage yielded similar results
[382]. Our laboratory has stored rabbit hippocampal slices under
isothermal conditions in the vicinity of À145
C (T G ~ À124
C)
for months as well, without detectable deterioration [248]. In Song
et al.’s experience, there was no visual development of ice during
storage, and freeze substitution showed no ice development after
5 months of storage in liquid nitrogen vapor [382]. Although
indirect, these observations are consistent with the possibility of
storing vitrified systems not far below T G .
Fahy and Rall [18] proposed that the kinetics of storage injury
at different temperatures might be predictable if biological deterioration is rate-limited by molecular motions and therefore by
increasing viscosity as temperature falls. Based on this assumption
and the relationship between T and viscosity in vitrifiable solutions,
the time t required for a given amount of deterioration to be
observed at storage temperature T can be related to the time t 1
required for the same amount of deterioration to be observed at a
higher reference temperature T 1 by the following equation:
t ¼ t 1 T 1 =T
ð
Þ exp B 1= T À T 0
ð
Þ
ð
ÞÀ1= T 1 À T 0
ð
Þ
½
½
,
where T 0 and B are empirical constants. The time required for
deterioration is not adjusted in this equation for the time required
for cooling to the storage temperature, but deterioration accumulated as a function of temperature during cooling at 1
C/min has
Principles of Vitrification
79