fundamental objectives for successful cryostorage of cells in liquid
nitrogen at À196
C can be summarized as follows: (1) arresting
the” metabolism reversibly, (2) maintaining structural and genetic
integrity, (3) achieving acceptable survival rates after thawing,
(4) maintaining the developmental competence post thaw, and
(5) making sure the technique has to be reliable and repeatable.
Cryopreservation slows or totally prevents unwanted physical
and chemical changes. The major disadvantage of using cryostorage
is that it can lead to crystallization of water and thereby can create
new and unwanted physical and chemical events that may injure the
cells that are being preserved. Vitrification, however, avoids ice
formation altogether during the cooling process by establishing a
glassy or vitreous state, wherein molecular translational motions are
arrested without structural reorganization of the liquid in which the
reproductive cells are suspended. To achieve this glass-like solidification of living cells for cryostorage, high cooling rates in the range
of 2500 to 30,000
C/min or greater in combination with high
concentrations of cryoprotectants are used. During vitrification
water is transformed directly from the liquid phase to a glassy
vitrified state. The physical definition of vitrification is the solidification of a solution at low temperature, not by ice crystallization
but by extreme elevation in viscosity during cooling [1, 2]. A primary strategy for vitrifying cells and tissue is to increase the speed of
thermal conductivity while decreasing the concentration of the
vitrificants to reduce their potential toxicity. In general, the rate of
cooling/warming and the concentration of the cryoprotectant
required to achieve vitrification are inversely related. In addition,
recent publications have shown the dominance of warming rate
over cooling rates in the survival of oocytes subjected to a vitrification procedure [3, 4].
The earliest attempts using vitrification as an ice-free cryopreservation method for embryos were first reported in 1985 [5]. In
1993 successful vitrification of mouse embryos was demonstrated
[6]. Furthermore, bovine oocytes and cleavage stages were vitrified
and warmed successfully a few years later [7]. In 1999 and 2000
successful pregnancies and deliveries after vitrification and warming
of human oocytes were reported [8, 9]. Since that time, and
because it seems to be that both entities appear to be especially
chill-sensitive cells in ART, oocytes and blastocysts seem to receive a
potentially significant boost in survival rates by avoiding ice crystallization using vitrification [10]. In general, vitrification solutions
are aqueous cryoprotectant solutions that do not freeze when
cooled at high cooling rates to very low temperature. Vitrification
is very simple, requires no expensive programmable freezing equipment, and relies especially on the placement of the embryo in a very
small volume of vitrification medium that must be cooled at
extreme rates not obtainable in traditional enclosed cryostorage
devices such as straws and vials.
502
Juergen Liebermann
nitrogen at À196
C can be summarized as follows: (1) arresting
the” metabolism reversibly, (2) maintaining structural and genetic
integrity, (3) achieving acceptable survival rates after thawing,
(4) maintaining the developmental competence post thaw, and
(5) making sure the technique has to be reliable and repeatable.
Cryopreservation slows or totally prevents unwanted physical
and chemical changes. The major disadvantage of using cryostorage
is that it can lead to crystallization of water and thereby can create
new and unwanted physical and chemical events that may injure the
cells that are being preserved. Vitrification, however, avoids ice
formation altogether during the cooling process by establishing a
glassy or vitreous state, wherein molecular translational motions are
arrested without structural reorganization of the liquid in which the
reproductive cells are suspended. To achieve this glass-like solidification of living cells for cryostorage, high cooling rates in the range
of 2500 to 30,000
C/min or greater in combination with high
concentrations of cryoprotectants are used. During vitrification
water is transformed directly from the liquid phase to a glassy
vitrified state. The physical definition of vitrification is the solidification of a solution at low temperature, not by ice crystallization
but by extreme elevation in viscosity during cooling [1, 2]. A primary strategy for vitrifying cells and tissue is to increase the speed of
thermal conductivity while decreasing the concentration of the
vitrificants to reduce their potential toxicity. In general, the rate of
cooling/warming and the concentration of the cryoprotectant
required to achieve vitrification are inversely related. In addition,
recent publications have shown the dominance of warming rate
over cooling rates in the survival of oocytes subjected to a vitrification procedure [3, 4].
The earliest attempts using vitrification as an ice-free cryopreservation method for embryos were first reported in 1985 [5]. In
1993 successful vitrification of mouse embryos was demonstrated
[6]. Furthermore, bovine oocytes and cleavage stages were vitrified
and warmed successfully a few years later [7]. In 1999 and 2000
successful pregnancies and deliveries after vitrification and warming
of human oocytes were reported [8, 9]. Since that time, and
because it seems to be that both entities appear to be especially
chill-sensitive cells in ART, oocytes and blastocysts seem to receive a
potentially significant boost in survival rates by avoiding ice crystallization using vitrification [10]. In general, vitrification solutions
are aqueous cryoprotectant solutions that do not freeze when
cooled at high cooling rates to very low temperature. Vitrification
is very simple, requires no expensive programmable freezing equipment, and relies especially on the placement of the embryo in a very
small volume of vitrification medium that must be cooled at
extreme rates not obtainable in traditional enclosed cryostorage
devices such as straws and vials.
502
Juergen Liebermann