membranes was observed [79]. Temperature reduction inhibits
most chemical reactions (e.g., alkaline phosphatase catalysis is
about 95% slower at À25
C than at 0
C [80]), and although
reactants can be concentrated greatly by the freezing process,
chemical reactions are generally not quickly driven forward as a
consequence, although enzymatic reaction rates may briefly
increase at high subzero temperatures in frozen model systems
[80], and formation of free radicals in conjunction with freezing
and thawing can induce sublethal injury in some cells [81]. Exothermic membrane liquid crystalline to gel phase transitions may be
favored during freezing, leading to cell membrane permeability
changes [84], but these phase changes do not destroy but only
rearrange the participating molecules and in most but not necessarily in all [85] cases are reversible. Freezing-induced cell shrinkage
can also drive lipid out of the cell membrane, after which it may or
may not be reincorporated upon thawing [86], but again, the lipid
itself is not chemically modified. Protein cold denaturation, discussed in more detail below, may or may not be spontaneously
reversible, but usually does not involve covalent modification of
the protein.
In some cases, the cryoprotectants used for vitrification may
inhibit and in some cases may promote covalent or non-covalent
changes in biomolecules, but their main purpose is to prevent
physical changes, particularly involving cell distortion, that precede
and thus are generally far more important than chemical reactions,
phase transitions, or protein denaturation for the survival of living
cells during cryopreservation.
1.4 Cryopreservation
by Vitrification: A
Conceptual History
A historical introduction to the field of cryopreservation by vitrification will help to put into perspective some of the key overall
concepts in approximately the order in which they were originally
developed. Additional reviews of the history of biological vitrification are available elsewhere [18–20, 49, 87–89].
1.4.1 Beginnings:
1930–1958
Cryopreservation by vitrification was apparently first introduced
conceptually although without either clarity or any empirical evidence by Stiles [90] in 1930 (quoted in [18]). Apparently independently, the idea was reintroduced much more clearly and
influentially by Luyet [7] in 1937. Both Stiles and Luyet were
inspired in part by Tamman’s finding that 38% of tested organic
compounds could be vitrified by rapid cooling [91] and in part by
indications that aqueous gelatin gels could be vitrified
[92, 93]. The concept was that if the water in living systems
could be cooled rapidly enough, there would be insufficient time
for crystals to form before reaching the glass transition temperature
of water, and the living system could therefore be trapped in the
vitreous state [7]. Luyet extensively described the concepts of
vitrification and devitrification and cited indications that quenched
Principles of Vitrification
33
most chemical reactions (e.g., alkaline phosphatase catalysis is
about 95% slower at À25
C than at 0
C [80]), and although
reactants can be concentrated greatly by the freezing process,
chemical reactions are generally not quickly driven forward as a
consequence, although enzymatic reaction rates may briefly
increase at high subzero temperatures in frozen model systems
[80], and formation of free radicals in conjunction with freezing
and thawing can induce sublethal injury in some cells [81]. Exothermic membrane liquid crystalline to gel phase transitions may be
favored during freezing, leading to cell membrane permeability
changes [84], but these phase changes do not destroy but only
rearrange the participating molecules and in most but not necessarily in all [85] cases are reversible. Freezing-induced cell shrinkage
can also drive lipid out of the cell membrane, after which it may or
may not be reincorporated upon thawing [86], but again, the lipid
itself is not chemically modified. Protein cold denaturation, discussed in more detail below, may or may not be spontaneously
reversible, but usually does not involve covalent modification of
the protein.
In some cases, the cryoprotectants used for vitrification may
inhibit and in some cases may promote covalent or non-covalent
changes in biomolecules, but their main purpose is to prevent
physical changes, particularly involving cell distortion, that precede
and thus are generally far more important than chemical reactions,
phase transitions, or protein denaturation for the survival of living
cells during cryopreservation.
1.4 Cryopreservation
by Vitrification: A
Conceptual History
A historical introduction to the field of cryopreservation by vitrification will help to put into perspective some of the key overall
concepts in approximately the order in which they were originally
developed. Additional reviews of the history of biological vitrification are available elsewhere [18–20, 49, 87–89].
1.4.1 Beginnings:
1930–1958
Cryopreservation by vitrification was apparently first introduced
conceptually although without either clarity or any empirical evidence by Stiles [90] in 1930 (quoted in [18]). Apparently independently, the idea was reintroduced much more clearly and
influentially by Luyet [7] in 1937. Both Stiles and Luyet were
inspired in part by Tamman’s finding that 38% of tested organic
compounds could be vitrified by rapid cooling [91] and in part by
indications that aqueous gelatin gels could be vitrified
[92, 93]. The concept was that if the water in living systems
could be cooled rapidly enough, there would be insufficient time
for crystals to form before reaching the glass transition temperature
of water, and the living system could therefore be trapped in the
vitreous state [7]. Luyet extensively described the concepts of
vitrification and devitrification and cited indications that quenched
Principles of Vitrification
33