these two ends of the spectrum, there is an optimum cooling rate
that minimizes both sources of injury [157–161].
Unfortunately, the existence of an optimum cooling rate is
problematic. The optimum cooling rate can only be determined
experimentally for every cell type of interest, which is inconvenient,
particularly for multicellular tissues, which may contain not only
multiple cell types but also cells in different relationships to each
other and to the extracellular environment, all of which affects the
optimal cooling rate [158]. Moreover, the optimum cooling rate
depends in part on the permeability of the cell to water, which
varies from cell type to cell type. Given the existence of a different
optimal cooling rate for different cells, finding a compromise rate
that gives high recoveries of all cells may be difficult and has been
proposed as a limiting factor for cryopreserving complex systems
[158]. Furthermore, even for a given type of cell, the optimum
cooling rate very often fails to yield 100% cell survival
[158, 162]. Finally, the use of cryoprotective agents to increase
survival at the optimal cooling rate also changes the optimal cooling
rate itself [162], again in a way that will be cell type dependent. For
these reasons, vitrification is advantageous in part because it transcends the need to find an optimal cooling rate, to compromise the
survival of one cell type to ensure survival of another cell type, and
to accept cell survival rates that are unlikely to approach 100%.
As noted, pCPAs must generally be used to obtain high survival
after freezing and thawing, since they mitigate solutions effects
injury. Although relatively low concentrations of pCPAs are needed
to prevent solution effects injury in many cells, the concentrating
effect of freezing on dissolved solutes pertains just as much to
pCPAs as it does to other solutes, the result being that pCPA
concentrations may be driven high enough in the frozen state to
induce toxic effects of their own [47, 163–165]. Interestingly, the
concentrations generated by freezing actually exceed the concentrations required for the vitrification of even large living systems
[105, 133, 166], so the advantage of using lower concentrations
for freezing is not necessarily as large as it at first appears.
Vitrification has an advantage over freezing also because some
important living systems such as oocytes are subject to chilling
injury (see below), and attempts to cool more rapidly than the
kinetics of chilling injury are precluded if the result is death secondary to IIF (intracellular ice formation). Vitrification eliminates that
obstacle by eliminating IIF at high cooling rates and has often been
pursued for that reason [64, 66, 145, 167].
Beyond changes in solution composition and IIF, freezing can
result in injury in at least two additional ways, both of them
mechanical in nature. First, the physical displacement of structures
in organized tissues by the simple growth of extracellular ice can
cause considerable damage to both the vascular bed and to parenchymal structures [130, 168–177]. In fact, it was the observation
40
Gregory M. Fahy and Brian Wowk
that minimizes both sources of injury [157–161].
Unfortunately, the existence of an optimum cooling rate is
problematic. The optimum cooling rate can only be determined
experimentally for every cell type of interest, which is inconvenient,
particularly for multicellular tissues, which may contain not only
multiple cell types but also cells in different relationships to each
other and to the extracellular environment, all of which affects the
optimal cooling rate [158]. Moreover, the optimum cooling rate
depends in part on the permeability of the cell to water, which
varies from cell type to cell type. Given the existence of a different
optimal cooling rate for different cells, finding a compromise rate
that gives high recoveries of all cells may be difficult and has been
proposed as a limiting factor for cryopreserving complex systems
[158]. Furthermore, even for a given type of cell, the optimum
cooling rate very often fails to yield 100% cell survival
[158, 162]. Finally, the use of cryoprotective agents to increase
survival at the optimal cooling rate also changes the optimal cooling
rate itself [162], again in a way that will be cell type dependent. For
these reasons, vitrification is advantageous in part because it transcends the need to find an optimal cooling rate, to compromise the
survival of one cell type to ensure survival of another cell type, and
to accept cell survival rates that are unlikely to approach 100%.
As noted, pCPAs must generally be used to obtain high survival
after freezing and thawing, since they mitigate solutions effects
injury. Although relatively low concentrations of pCPAs are needed
to prevent solution effects injury in many cells, the concentrating
effect of freezing on dissolved solutes pertains just as much to
pCPAs as it does to other solutes, the result being that pCPA
concentrations may be driven high enough in the frozen state to
induce toxic effects of their own [47, 163–165]. Interestingly, the
concentrations generated by freezing actually exceed the concentrations required for the vitrification of even large living systems
[105, 133, 166], so the advantage of using lower concentrations
for freezing is not necessarily as large as it at first appears.
Vitrification has an advantage over freezing also because some
important living systems such as oocytes are subject to chilling
injury (see below), and attempts to cool more rapidly than the
kinetics of chilling injury are precluded if the result is death secondary to IIF (intracellular ice formation). Vitrification eliminates that
obstacle by eliminating IIF at high cooling rates and has often been
pursued for that reason [64, 66, 145, 167].
Beyond changes in solution composition and IIF, freezing can
result in injury in at least two additional ways, both of them
mechanical in nature. First, the physical displacement of structures
in organized tissues by the simple growth of extracellular ice can
cause considerable damage to both the vascular bed and to parenchymal structures [130, 168–177]. In fact, it was the observation
40
Gregory M. Fahy and Brian Wowk