shown to undergo a glass transition at about À45
C and are known
to be able to survive immersion in liquid nitrogen [195]. Highly
frost-hardy plants in general, according to Sakai, survive in conjunction with their ability to form intracellular glasses [144].
1.7 Vitrification
During Freezing
of Living Cells
As described above, when ice forms in the presence of cryoprotectants, their concentrations are elevated by loss of water from the
solution into the ice phase, until eventually they preclude further
ice formation during continued cooling, resulting ultimately in
vitrification of the residual unfrozen solution [105, 110] (see also
Subheadings 1.4 and 2.1). Similarly, freeze concentration of the
extracellular solution and concomitant osmotic reduction of cell
volume (see also Subheading 1.5) results in vitrification of cytoplasm when cooling is slow enough to preclude IIF [105, 139–141,
196]. Therefore, most cells survive cryopreservation as a result of
vitrification, even if the medium surrounding them is not
completely vitrified.
2 The Physical Principles of Vitrification
2.1 Vitrification
Depends on the Solute
Concentration
of Aqueous Solutions
Figure 2 illustrates the process described in Subheading 1.7,
showing the example of the slow freezing of a 10% glycerol solution
in water on a glycerol-water phase diagram. The solution may
initially supercool before the first ice crystal forms, but thereafter
the concentration of the remaining unfrozen solution follows the
melting temperature line (T m ) with continued cooling. Increasing
solution viscosity during cooling eventually inhibits ice growth,
causing a small departure from thermodynamic equilibrium
[29]. Final cooling then continues with little change in concentration until the glass transition temperature (T G ) is reached
[105]. Below T G , the viscosity of the unfrozen solution between
ice crystals becomes so high (>10
13 poise) that the solution
behaves as a solid. However, it does so while retaining the random
molecular arrangement of a liquid. As noted above, a solid with the
same unstructured molecular arrangements as a liquid is called a
glass [1].
With a sufficiently high solute concentration and/or cooling
rate, it is possible to cool all the way to the glass transition temperature without significant ice formation [105, 133]. This is the basis
of cryopreservation by vitrification. During cryopreservation by
vitrification, the entire sample volume remains substantially free of
ice during cooling. As shown in Fig. 3, this can be achieved by using
either low solute concentrations and fast cooling rates or higher
concentrations and slower cooling rates. Heat transfer limitations
necessitate the use of high solute concentration and slow cooling
rates when vitrifying large volumes, such as tissues and organs.
Principles of Vitrification
43
C and are known
to be able to survive immersion in liquid nitrogen [195]. Highly
frost-hardy plants in general, according to Sakai, survive in conjunction with their ability to form intracellular glasses [144].
1.7 Vitrification
During Freezing
of Living Cells
As described above, when ice forms in the presence of cryoprotectants, their concentrations are elevated by loss of water from the
solution into the ice phase, until eventually they preclude further
ice formation during continued cooling, resulting ultimately in
vitrification of the residual unfrozen solution [105, 110] (see also
Subheadings 1.4 and 2.1). Similarly, freeze concentration of the
extracellular solution and concomitant osmotic reduction of cell
volume (see also Subheading 1.5) results in vitrification of cytoplasm when cooling is slow enough to preclude IIF [105, 139–141,
196]. Therefore, most cells survive cryopreservation as a result of
vitrification, even if the medium surrounding them is not
completely vitrified.
2 The Physical Principles of Vitrification
2.1 Vitrification
Depends on the Solute
Concentration
of Aqueous Solutions
Figure 2 illustrates the process described in Subheading 1.7,
showing the example of the slow freezing of a 10% glycerol solution
in water on a glycerol-water phase diagram. The solution may
initially supercool before the first ice crystal forms, but thereafter
the concentration of the remaining unfrozen solution follows the
melting temperature line (T m ) with continued cooling. Increasing
solution viscosity during cooling eventually inhibits ice growth,
causing a small departure from thermodynamic equilibrium
[29]. Final cooling then continues with little change in concentration until the glass transition temperature (T G ) is reached
[105]. Below T G , the viscosity of the unfrozen solution between
ice crystals becomes so high (>10
13 poise) that the solution
behaves as a solid. However, it does so while retaining the random
molecular arrangement of a liquid. As noted above, a solid with the
same unstructured molecular arrangements as a liquid is called a
glass [1].
With a sufficiently high solute concentration and/or cooling
rate, it is possible to cool all the way to the glass transition temperature without significant ice formation [105, 133]. This is the basis
of cryopreservation by vitrification. During cryopreservation by
vitrification, the entire sample volume remains substantially free of
ice during cooling. As shown in Fig. 3, this can be achieved by using
either low solute concentrations and fast cooling rates or higher
concentrations and slower cooling rates. Heat transfer limitations
necessitate the use of high solute concentration and slow cooling
rates when vitrifying large volumes, such as tissues and organs.
Principles of Vitrification
43