(lethal) intracellular ice formation [26]. The ice nucleation temperature determines the extent of supercooling and thereby the extent
of cellular dehydration and likelihood of intracellular ice
formation [25].
The two-factor hypothesis explains cryosurvival with slow cooling cryopreservation protocols (see Fig. 5a). At cooling rates below
the optimal cooling rate, cell death can be attributed to the deleterious effects of exposure to high salt concentrations in the unfrozen
fraction, whereas at cooling rates greater than the optimal cooling
rate, survival drops because intracellular freezing formation supervenes. It has been postulated that each cell has an optimum cooling
rate [18, 26]. Controlled rate freezing determines the extent of
dehydration during freezing, which allows cells to survive freezing.
It has been postulated that the optimal cooling rate resulting in
maximal cryosurvival coincides with the rate where the osmotically
active cell volume has reduced down to 90–95% of the original
volume at À30
C [59].
In the presence of CPAs, optimal survival is often found over a
broad range of cooling rates rather than at a defined cooling rate.
Optimal cooling rates differ dependent on the type and concentration of CPA that is used, and cryosurvival is determined by a
complex interplay between the type and concentration of CPA,
and the cooling velocity [76, 96]. At the right combination of
cooling rate and CPA concentration, many CPAs may turn out to
be equally effective.
6.3 Cryopreservation
by Vitrification
The damaging effects of ice formation can be avoided using vitrification or ice-free cryopreservation, which is typically preferred for
embryo and tissues. With vitrification procedures, there is no need
to know the specific optimal rate where cell survival is maximal
making it inherently more suitable to preserve specimens consisting
of multiple cell types. Vitrification is done using high CPA concentrations and high cooling rates, allowing samples to directly turn
into an amorphous state protecting embedded structures while
arresting chemical reactions [19, 97]. Typically, mixtures of permeating agents and step-wise CPA loading and removal protocols
are used to reduce CPA toxicity and remain within the osmotic
tolerance limits [63, 98]. Vitrification is used for mammalian as well
as plant tissues, but its application is mostly limited to smaller tissue
pieces. Vitrification of larger tissues is challenging due to limitations in homogeneous CPA mass transfer and inhomogeneous heat
transfer during rapid cooling and warming. Application of very
high CPA concentrations allows using lower cooling rates. However, this causes a decline in cell viability already prior to cooling
due to CPA toxicity [99].
Vitrification solutions typically consist of CPA mixtures to
avoid CPA toxicity toward one of the components. Moreover,
this reduces the risk of solute crystallization, which is more likely
18
Willem F. Wolkers and Harrie ¨ tte Oldenhof
of cellular dehydration and likelihood of intracellular ice
formation [25].
The two-factor hypothesis explains cryosurvival with slow cooling cryopreservation protocols (see Fig. 5a). At cooling rates below
the optimal cooling rate, cell death can be attributed to the deleterious effects of exposure to high salt concentrations in the unfrozen
fraction, whereas at cooling rates greater than the optimal cooling
rate, survival drops because intracellular freezing formation supervenes. It has been postulated that each cell has an optimum cooling
rate [18, 26]. Controlled rate freezing determines the extent of
dehydration during freezing, which allows cells to survive freezing.
It has been postulated that the optimal cooling rate resulting in
maximal cryosurvival coincides with the rate where the osmotically
active cell volume has reduced down to 90–95% of the original
volume at À30
C [59].
In the presence of CPAs, optimal survival is often found over a
broad range of cooling rates rather than at a defined cooling rate.
Optimal cooling rates differ dependent on the type and concentration of CPA that is used, and cryosurvival is determined by a
complex interplay between the type and concentration of CPA,
and the cooling velocity [76, 96]. At the right combination of
cooling rate and CPA concentration, many CPAs may turn out to
be equally effective.
6.3 Cryopreservation
by Vitrification
The damaging effects of ice formation can be avoided using vitrification or ice-free cryopreservation, which is typically preferred for
embryo and tissues. With vitrification procedures, there is no need
to know the specific optimal rate where cell survival is maximal
making it inherently more suitable to preserve specimens consisting
of multiple cell types. Vitrification is done using high CPA concentrations and high cooling rates, allowing samples to directly turn
into an amorphous state protecting embedded structures while
arresting chemical reactions [19, 97]. Typically, mixtures of permeating agents and step-wise CPA loading and removal protocols
are used to reduce CPA toxicity and remain within the osmotic
tolerance limits [63, 98]. Vitrification is used for mammalian as well
as plant tissues, but its application is mostly limited to smaller tissue
pieces. Vitrification of larger tissues is challenging due to limitations in homogeneous CPA mass transfer and inhomogeneous heat
transfer during rapid cooling and warming. Application of very
high CPA concentrations allows using lower cooling rates. However, this causes a decline in cell viability already prior to cooling
due to CPA toxicity [99].
Vitrification solutions typically consist of CPA mixtures to
avoid CPA toxicity toward one of the components. Moreover,
this reduces the risk of solute crystallization, which is more likely
18
Willem F. Wolkers and Harrie ¨ tte Oldenhof