about 65 mole percent of its side chains replaced with carboxyl
groups, has also shown recrystallization inhibition and good cryopreservation by freezing [252, 253] as well as successful applications to vitrification [254–257]. Certain polyampholytes (polymers
with mixed cationic and anionic groups) have also been found to be
IRIs and effective cryoprotectants [252]. Some new synthetic IRI
molecules are small enough to enter cells and inhibit recrystallization intracellularly [56, 57].
2.7 Thermally
Induced Volume
Changes, Strain,
and Fracture
Formation
Like most matter, cryoprotectant solutions contract with cooling,
possessing a linear thermal expansion coefficient of ~90 ppm/
C
[258]. Below the glass transition temperature, the thermal expansion coefficient is observed to fall to ~40 ppm/
C [258] due to
kinetic inhibition of molecular movements required to continue
liquid behavior. This causes mechanical stress in samples during
vitrification. Colder outer parts of a sample will solidify (take on a
lower thermal expansion coefficient) before warmer inner parts of a
sample do. The resulting tendency of the interior to contract more
than the exterior after passage of the latter through the glass
transition and subsequent approach to thermal equilibrium creates
stress. Cryoprotectant-water glasses have a fracture strain of ~0.3%
and fracture stress of ~3 MPa [259], which is only one tenth that of
silica glass (due to hydrogen bonding rather than covalent bonding
in aqueous cryoprotectant glasses). This makes samples prone to
fracturing during vitrification [109, 180, 182]. Fracturing is undesirable because fracture planes nucleate ice [182] and unacceptably
alter tissues [260], organs, and even cellular systems [181].
Stress during cooling to temperatures far below T G has been
found to be proportional to the cooling rate and the square of the
linear sample size [261]. It is best managed by slowing the cooling
rate as T G is approached [180] and ensuring sample temperature
uniformity as best as reasonably possible during passage below T G .
Ensuring that samples don’t adhere to container surfaces during cooling is especially important for fracture avoidance [88]
because containers typically respond differently to cooling than
cryoprotectant solutions. Hydrophobic polymers that permit sample retraction away from container walls during cooling are to be
preferred over hydrophilic materials such as borosilicate glass.
3 The Biological Principles of Vitrification
3.1 Are
Cryoprotectants
Necessary
for Vitrification?
Cryopreservation by vitrification without added cryoprotectants
has been a goal of investigators at least since the introduction of
the idea of vitrification in the 1930s. It has been known for many
years that very small biological systems and even pure water can be
cooled rapidly enough to avoid ice crystals that are visible in the
electron microscope [10], but that is a different problem than
Principles of Vitrification
55
groups, has also shown recrystallization inhibition and good cryopreservation by freezing [252, 253] as well as successful applications to vitrification [254–257]. Certain polyampholytes (polymers
with mixed cationic and anionic groups) have also been found to be
IRIs and effective cryoprotectants [252]. Some new synthetic IRI
molecules are small enough to enter cells and inhibit recrystallization intracellularly [56, 57].
2.7 Thermally
Induced Volume
Changes, Strain,
and Fracture
Formation
Like most matter, cryoprotectant solutions contract with cooling,
possessing a linear thermal expansion coefficient of ~90 ppm/
C
[258]. Below the glass transition temperature, the thermal expansion coefficient is observed to fall to ~40 ppm/
C [258] due to
kinetic inhibition of molecular movements required to continue
liquid behavior. This causes mechanical stress in samples during
vitrification. Colder outer parts of a sample will solidify (take on a
lower thermal expansion coefficient) before warmer inner parts of a
sample do. The resulting tendency of the interior to contract more
than the exterior after passage of the latter through the glass
transition and subsequent approach to thermal equilibrium creates
stress. Cryoprotectant-water glasses have a fracture strain of ~0.3%
and fracture stress of ~3 MPa [259], which is only one tenth that of
silica glass (due to hydrogen bonding rather than covalent bonding
in aqueous cryoprotectant glasses). This makes samples prone to
fracturing during vitrification [109, 180, 182]. Fracturing is undesirable because fracture planes nucleate ice [182] and unacceptably
alter tissues [260], organs, and even cellular systems [181].
Stress during cooling to temperatures far below T G has been
found to be proportional to the cooling rate and the square of the
linear sample size [261]. It is best managed by slowing the cooling
rate as T G is approached [180] and ensuring sample temperature
uniformity as best as reasonably possible during passage below T G .
Ensuring that samples don’t adhere to container surfaces during cooling is especially important for fracture avoidance [88]
because containers typically respond differently to cooling than
cryoprotectant solutions. Hydrophobic polymers that permit sample retraction away from container walls during cooling are to be
preferred over hydrophilic materials such as borosilicate glass.
3 The Biological Principles of Vitrification
3.1 Are
Cryoprotectants
Necessary
for Vitrification?
Cryopreservation by vitrification without added cryoprotectants
has been a goal of investigators at least since the introduction of
the idea of vitrification in the 1930s. It has been known for many
years that very small biological systems and even pure water can be
cooled rapidly enough to avoid ice crystals that are visible in the
electron microscope [10], but that is a different problem than
Principles of Vitrification
55