that dog kidneys frozen to À30
C and stored for a week using 3 M
glycerol could perfuse normally and respond well to pressors
in vitro but produced urine consisting largely of whole blood and
stopped perfusing within 10–20 min of being transplanted
(G. Fahy, M. Goldman, and H. T. Meryman, unpublished results)
that inspired the proposal to investigate vitrification as a more
promising approach to organ cryopreservation. Fortunately, Taylor
and his colleagues have provided extensive microscopic evidence
using freeze substitution methods that vitrifiable solutions successfully prevent tissue distortion by ice [19, 147, 178]. Second, even
single cells can be injured by intracellular [158] and extracellular
[58, 172, 173] recrystallization. Both forms of mechanical injury
secondary to ice formation argue for vitrification as a potentially less
damaging preservation method, particularly for complex organized
tissues and organs.
Vitrification does have significant disadvantages as well, however [146, 179]. First, the need to tolerate very high concentrations
of CPA requires relatively sophisticated methods of adding and
removing these agents and careful selection of the right CPA
blend for the living system at hand. Second, it is not always clear
what CPA concentration and exposure time are needed to ensure
vitrification and maintenance of an ice-free state upon warming in
specific cases, and investigation of these points may be needed.
Third, rapid cooling to below the glass transition temperature
and/or rapid warming below T G may induce fracturing of the
glass in which the biological system is embedded [109, 180] (see
also Subheading 2.7), which may cleave cells or tissues, causing
irreversible injury [181] and additional ice nucleation
[182]. Fourth, to avoid the third problem, in some cases storage
at “intermediate temperatures” [~À130 to À160
C, i.e., below T G
but above the temperature of boiling liquid nitrogen (À196
C)]
may be needed to ensure long-term preservation without fracturing
(see also Subheading 3.1). Finally, although rapid warming is generally beneficial for frozen systems, it can be even more important
for vitrified ones due to the need to avoid injury from devitrification
and subsequent recrystallization.
A potential disadvantage of certain techniques of vitrification
comes from the use of container-free cooling methods, which are
intended to accelerate cooling and warming rates and thereby
enable the use of minimal concentrations of cryoprotectant but
may result in contamination of the sample being preserved
[146]. The need for such methods is questionable, however, and
it seems likely that closed-system vitrification will eventually remove
the risk of contamination [183].
1.6 Vitrification
in Nature
It is reassuring that nature has often drawn the same conclusion as
the cryobiologist in pursuing ice-free cryopreservation in preference to freezing.
Principles of Vitrification
41
C and stored for a week using 3 M
glycerol could perfuse normally and respond well to pressors
in vitro but produced urine consisting largely of whole blood and
stopped perfusing within 10–20 min of being transplanted
(G. Fahy, M. Goldman, and H. T. Meryman, unpublished results)
that inspired the proposal to investigate vitrification as a more
promising approach to organ cryopreservation. Fortunately, Taylor
and his colleagues have provided extensive microscopic evidence
using freeze substitution methods that vitrifiable solutions successfully prevent tissue distortion by ice [19, 147, 178]. Second, even
single cells can be injured by intracellular [158] and extracellular
[58, 172, 173] recrystallization. Both forms of mechanical injury
secondary to ice formation argue for vitrification as a potentially less
damaging preservation method, particularly for complex organized
tissues and organs.
Vitrification does have significant disadvantages as well, however [146, 179]. First, the need to tolerate very high concentrations
of CPA requires relatively sophisticated methods of adding and
removing these agents and careful selection of the right CPA
blend for the living system at hand. Second, it is not always clear
what CPA concentration and exposure time are needed to ensure
vitrification and maintenance of an ice-free state upon warming in
specific cases, and investigation of these points may be needed.
Third, rapid cooling to below the glass transition temperature
and/or rapid warming below T G may induce fracturing of the
glass in which the biological system is embedded [109, 180] (see
also Subheading 2.7), which may cleave cells or tissues, causing
irreversible injury [181] and additional ice nucleation
[182]. Fourth, to avoid the third problem, in some cases storage
at “intermediate temperatures” [~À130 to À160
C, i.e., below T G
but above the temperature of boiling liquid nitrogen (À196
C)]
may be needed to ensure long-term preservation without fracturing
(see also Subheading 3.1). Finally, although rapid warming is generally beneficial for frozen systems, it can be even more important
for vitrified ones due to the need to avoid injury from devitrification
and subsequent recrystallization.
A potential disadvantage of certain techniques of vitrification
comes from the use of container-free cooling methods, which are
intended to accelerate cooling and warming rates and thereby
enable the use of minimal concentrations of cryoprotectant but
may result in contamination of the sample being preserved
[146]. The need for such methods is questionable, however, and
it seems likely that closed-system vitrification will eventually remove
the risk of contamination [183].
1.6 Vitrification
in Nature
It is reassuring that nature has often drawn the same conclusion as
the cryobiologist in pursuing ice-free cryopreservation in preference to freezing.
Principles of Vitrification
41