Storage of unfertilized oocytes has numerous applications.
Originally conceived as a potential option for women undergoing
anticancer treatment which may render the women infertile [8], it
may now have a wider remit, including application in banking of
donated oocytes, premature ovarian failure, ethical objections to
embryo cryopreservation, or in women seeking to defer their
reproductive potential until later in life but who do not have a
current partner [4]. The method can also be used to cryobank
excess oocytes produced within a particular cycle during in vitro
fertilization.
Applications in animal management include the preservation of
important genetic lineage; the ability to restock following outbreaks of diseases such as the foot and mouth; the preservation of
genetically modified strains, thereby reducing the cost of continuous breeding and avoiding problems of genetic drift; and the
preservation of endangered species.
Oocyte cryopreservation has also developed alongside a greater
understanding of what is needed for successful recovery of function
after cryogenic storage. The need for optimized cryogenic dehydration of the cells, coupled with introduction of specific nontoxic
stabilizing solutes called cryoprotectants (CPA), is a central tenet of
cryobiology [2, 9, 10]. The term cryopreservation itself has come
to mean different things in applied cryobiology. For true long-term
cryogenic preservation, cells and their supporting medium need to
be stored in this optimally cryodehydrated state below the temperature range at which any residual water molecules undergo a “glassy
transition” [2, 11]. However, there are two different approaches to
achieve this optimally dehydrated state which can be termed controlled rate slow cooling (CRSC), or vitrification (VS—literally
changing into a glassy state). In CRSC CPAs are essential to support cell survival, but the dehydration is produced by the extracellular water within the sample being changed into ever-increasing
ice fraction as temperatures progress slowly (at rates < À1
C/min)
down from 0
C to À60
C and beyond. In VS CPAs are equally
essential, but they are applied in very high concentrations to produce optimal cryodehydration and suppress ice crystal formation
before cooling begins [2–4, 11]. Because these high CPA concentrations are potentially toxic, the samples must be cooled very
rapidly (at rates >200
C/min) to below the glassy transition
range (about À120
C, depending on the CPAs mixture selected).
VS as this is widely used can only be practically achieved using small
volume samples (usually <10 μL) in special containers to achieve
these high cooling rates. Lastly, for both CRSC and VS, the rate of
rewarming back from cryogenic storage is important to control,
and should be fast, to avoid the propensity for free water molecules
to form ice crystals as the temperature profile climbs (the so-called
event of freezing during warming) [12].
438
Victoria Keros and Barry J. Fuller
Originally conceived as a potential option for women undergoing
anticancer treatment which may render the women infertile [8], it
may now have a wider remit, including application in banking of
donated oocytes, premature ovarian failure, ethical objections to
embryo cryopreservation, or in women seeking to defer their
reproductive potential until later in life but who do not have a
current partner [4]. The method can also be used to cryobank
excess oocytes produced within a particular cycle during in vitro
fertilization.
Applications in animal management include the preservation of
important genetic lineage; the ability to restock following outbreaks of diseases such as the foot and mouth; the preservation of
genetically modified strains, thereby reducing the cost of continuous breeding and avoiding problems of genetic drift; and the
preservation of endangered species.
Oocyte cryopreservation has also developed alongside a greater
understanding of what is needed for successful recovery of function
after cryogenic storage. The need for optimized cryogenic dehydration of the cells, coupled with introduction of specific nontoxic
stabilizing solutes called cryoprotectants (CPA), is a central tenet of
cryobiology [2, 9, 10]. The term cryopreservation itself has come
to mean different things in applied cryobiology. For true long-term
cryogenic preservation, cells and their supporting medium need to
be stored in this optimally cryodehydrated state below the temperature range at which any residual water molecules undergo a “glassy
transition” [2, 11]. However, there are two different approaches to
achieve this optimally dehydrated state which can be termed controlled rate slow cooling (CRSC), or vitrification (VS—literally
changing into a glassy state). In CRSC CPAs are essential to support cell survival, but the dehydration is produced by the extracellular water within the sample being changed into ever-increasing
ice fraction as temperatures progress slowly (at rates < À1
C/min)
down from 0
C to À60
C and beyond. In VS CPAs are equally
essential, but they are applied in very high concentrations to produce optimal cryodehydration and suppress ice crystal formation
before cooling begins [2–4, 11]. Because these high CPA concentrations are potentially toxic, the samples must be cooled very
rapidly (at rates >200
C/min) to below the glassy transition
range (about À120
C, depending on the CPAs mixture selected).
VS as this is widely used can only be practically achieved using small
volume samples (usually <10 μL) in special containers to achieve
these high cooling rates. Lastly, for both CRSC and VS, the rate of
rewarming back from cryogenic storage is important to control,
and should be fast, to avoid the propensity for free water molecules
to form ice crystals as the temperature profile climbs (the so-called
event of freezing during warming) [12].
438
Victoria Keros and Barry J. Fuller