minimal concentrations of cryoprotectant and maximal cooling and
warming rates [146], more akin to the original proposals of Boutron and of James and Farrant. These methods remain useful and
continue to be refined to the present time, although they blur the
distinction between true vitrification and rapid freezing (see Subheading 3.2).
In the year 2000, the first evidence came to light for the
superiority of vitrification over freezing for organized mammalian
tissues (in this case, rabbit jugular veins) [147], and the first evidence was published showing that a mammalian organ can survive
after perfusion with a vitrifiable solution followed by transplantation [148]. Survival of a vital mammalian organ after vitrification
and transplantation was reported in 2009 [22]. Today, ice-free
cryopreservation is receiving increasing attention, and many new
avenues are being investigated.
1.5 Advantages
and Disadvantages
of Vitrification
The overall purpose of vitrification is to achieve cryopreservation
while avoiding freezing. To understand the advantages and disadvantages of vitrification, it is therefore necessary to understand
something about freezing injury.
Conventional cryopreservation by freezing involves, by definition, the formation and dissolution of ice during cooling and
warming, respectively. Ice is an almost completely pure substance,
so its formation subtracts solvent water from a freezing solution,
leaving the dissolved solutes in a reduced volume of solvent. The
effects of ice formation are in part due to this concentrating action,
which increases both the osmotic concentration of the cellular
environment and the individual concentrations of dissolved solutes
such as electrolytes, buffers, etc. [149, 150]. If cooling proceeds
sufficiently slowly, ice formation begins extracellularly [151, 152],
there is time for cells to lose water down the transmembrane
osmotic gradient established by the extracellular ice, and the cells
will consequently shrink. If shrinkage proceeds too far, osmotic
injury may result [153–156]. If cooling proceeds more rapidly,
the rate of water subtraction from the cell fails to keep up with
the rate of water subtraction from the extracellular environment,
leaving the cell interior significantly more dilute than the extracellular solution [157]. This means that the thermodynamic freezing
point of the cell fails to fall as rapidly as the prevailing temperature,
i.e., that the temperature of the cytosol begins to fall farther and
farther below its nominal freezing point. This defines a state of
supercooling (cooling below the freezing point without ice formation), and as supercooling increases, the risk of ice formation within
the cytosol increases.
In summary, cells cooled too slowly are liable to injury related
to shrinkage and changes in solution composition (“solution
effects” injury), whereas cells cooled too quickly are liable to injury
related to intracellular ice formation (IIF) [157–161]. Between
Principles of Vitrification
39
warming rates [146], more akin to the original proposals of Boutron and of James and Farrant. These methods remain useful and
continue to be refined to the present time, although they blur the
distinction between true vitrification and rapid freezing (see Subheading 3.2).
In the year 2000, the first evidence came to light for the
superiority of vitrification over freezing for organized mammalian
tissues (in this case, rabbit jugular veins) [147], and the first evidence was published showing that a mammalian organ can survive
after perfusion with a vitrifiable solution followed by transplantation [148]. Survival of a vital mammalian organ after vitrification
and transplantation was reported in 2009 [22]. Today, ice-free
cryopreservation is receiving increasing attention, and many new
avenues are being investigated.
1.5 Advantages
and Disadvantages
of Vitrification
The overall purpose of vitrification is to achieve cryopreservation
while avoiding freezing. To understand the advantages and disadvantages of vitrification, it is therefore necessary to understand
something about freezing injury.
Conventional cryopreservation by freezing involves, by definition, the formation and dissolution of ice during cooling and
warming, respectively. Ice is an almost completely pure substance,
so its formation subtracts solvent water from a freezing solution,
leaving the dissolved solutes in a reduced volume of solvent. The
effects of ice formation are in part due to this concentrating action,
which increases both the osmotic concentration of the cellular
environment and the individual concentrations of dissolved solutes
such as electrolytes, buffers, etc. [149, 150]. If cooling proceeds
sufficiently slowly, ice formation begins extracellularly [151, 152],
there is time for cells to lose water down the transmembrane
osmotic gradient established by the extracellular ice, and the cells
will consequently shrink. If shrinkage proceeds too far, osmotic
injury may result [153–156]. If cooling proceeds more rapidly,
the rate of water subtraction from the cell fails to keep up with
the rate of water subtraction from the extracellular environment,
leaving the cell interior significantly more dilute than the extracellular solution [157]. This means that the thermodynamic freezing
point of the cell fails to fall as rapidly as the prevailing temperature,
i.e., that the temperature of the cytosol begins to fall farther and
farther below its nominal freezing point. This defines a state of
supercooling (cooling below the freezing point without ice formation), and as supercooling increases, the risk of ice formation within
the cytosol increases.
In summary, cells cooled too slowly are liable to injury related
to shrinkage and changes in solution composition (“solution
effects” injury), whereas cells cooled too quickly are liable to injury
related to intracellular ice formation (IIF) [157–161]. Between
Principles of Vitrification
39