For more than 20 years, scientists have had the capability to successfully freeze and retrieve viable mouse embryos. These living embryos may be stored for a conceivably infinite period of time and then used to regenerate a colony of live animals. Embryos stored at liquid nitrogen temperature (-196°C) are inactive metabolically. Molecular activities, especially enzyme function, and DNA mutation, do not occur.
While scientists have been able to freeze the sperm of other mammalian species, freezing mouse sperm for storage and later use in producing a viable embryo has proven difficult. These problems in cryopreservation of mouse sperm add to the significance of instead freezing mouse embryos.
Several advantages of maintaining potentially valuable animal colonies in frozen storage are apparent:
The era of successful embryo cryopreservation followed the discovery and development of chemical cryoprotectant compounds. Interestingly, the original discovery of the value of cryoprotectants was accomplished by accident. In 1949, Christopher Polge in the United Kingdom inadvertently supplemented an experimental freezing solution with glycerol, resulting in the unexpected survival of his experimentally frozen cells. By the early 1970's, Ian Wilmut and Whittingham (also in the UK) had developed independent methods for reliably freezing mouse embryos in dimethylsulfoxide (DMSO). Currently, the most popular mouse embryo cryoprotectant solutions include DMSO, ethylene glycol, propylene glycol (1-2, propanediol), and glycerol. (Glycerol is the active ingredient in auto antifreeze solutions.) Cryoprotectant solutions frequently are supplemented with glucose or sucrose to enhance freezing and thawing osmotic equilibration phenomena. By the 1980's, the freezing of human embryos had emerged as a common procedure in clinical infertility centers.
Cryoprotective agents like DMSO and glycerol improve the survival rate of frozen cells by decreasing the temperature at which ice forms. The movement of water into and out of the cell during chilling determines the dynamics of intracellular ice formation and cell survival.
The most commonly frozen stages of mouse embryos are the 2-cell and pre-compaction 8-cell stages. Large numbers of synchronously timed embryos may be collected either two days post-coitus (2-cell) or three days p.c. (8-cell). These early preimplantation embryos are undifferentiated and contain presumably totipotent blastomeres (the cells of the preimplantation embryo). Therefore, survival of the embryo may occur even if 50% or even 85% of the embryo's cells are killed. Pronuclear and 1-cell stage embryos also may be frozen successfully, but their delicate and dynamic cytoskeletal and nuclear structures are more prone to freezing damage. The freezing and thawing of the complex blastocyst stage embryo also presents a more difficult challenge, due to the dynamics of the fluid-filled blastocoele cavity. Regardless of developmental stage, only embryos with intact cell membranes and zona pellucidas are expected to survive freezing and thawing.
Water is the major component of living cells and the physical structure of water responds predictably to changes in temperature. Decreased temperature eventually yields the development of intracellular ice crystals, which will damage the plasma membrane and organelles of living cells. Living cells may be frozen successfully if the freezing medium is supplemented with a cryoprotectant compound.
Mouse embryos are frozen using relatively low molecular weight, permeable cryoprotectant compounds. The term permeable refers to the ability of the agent to enter living cells at temperatures above 0°C. A relatively brief period of cellular equilibration (in the range of 25°C to 0°C), within the cryoprotectant solution, usually is required prior to initiation of the freezing procedure. These agents are highly water-soluble and almost exclusively employed in the concentration range of 0.5 to 2.0 M. Cryoprotectants lower the freezing point of the cell (to -4 to -6°C), thereby permitting the controlled dehydration of the cells. If performed properly, controlled cryopreservation regulates the intracellular concentration of electrolytes so that cell viability is not compromised.
The rapidity of the freezing process must be controlled. The optimal rate of cooling is dependent on the surface-to-volume ratio and water permeability of the cell. Mouse embryos almost always are frozen at a rate in the chilling range of 0.2 to 1.0°C per minute. This programmed, slow freezing strategy ensures that ice will form surrounding the zona pellucidas of the embryos, rather than within the embryonic cells or blastomeres.
Immediately prior to the initiation of the freezing program, this extracellular fluid is cooled to approximately -5° to -7°C and induced to freeze by a process referred to as seeding. The seeding temperature chosen should be just below the freezing point of the extracellular medium. Once the extracellular water freezes, its vapor pressure is lower than the unfrozen intracellular water. When the extracellular water changes from liquid to ice, the solutes become more concentrated. The result is a net flow of water out of the cell, to restore equilibrium across the cell membrane.
The critical process of seeding prevents the potentially damaging effects of an interesting cryobiological phenomenon. Normally, as ice crystals form within the medium, the resulting latent heat of crystallization would cause the temperature of the sample to rise back to the freezing point, whereas the temperature of the freezing bath would decrease to the much lower, programmed temperature. The cells would then cool at a very rapid rate, irrespective of the programmed cooling rate. Seeding to induce extracellular ice formation prevents the occurrence of this scenario.
At an appropriate rate of cooling in the presence of a cryoprotectant compound, the cell will shrink and the formation of intracellular ice will be avoided. If cooling is too rapid, a large of amount of intracellular ice will form. If the rate of cooling is much slower than the optimum, cell damage may result from prolonged exposure to toxic solute concentrations. The cells are frozen to a target temperature of -40 to -80°C and immediately placed into storage in liquid nitrogen (-196°C).
The thawing procedure for the frozen cells depends on the specific freezing protocol and choice of cryoprotectant. For mouse embryos that were slowly frozen, rapid warming from the -196°C storage temperature is the rule. Frozen embryos may be warmed to room temperature (at a warming pace of 10° to 20°C per minute) or to 37°C (at a rapid pace of several hundred degrees C per minute).
The intracellular cryoprotectant compounds must be removed from the cells by immersion of the thawed sample in DPBS or M2 solution. Frequently, stepwise dilution and cryoprotectant removal is performed, rather than direct dilution. The embryos should swell slightly and appear normal within 20 minutes after thawing.
A face safety shield, lab coat, and low-temperature protective gloves should be worn when handling vials or racks in a liquid nitrogen freezer. Improperly sealed vials can explode, producing dangerous projectiles of glass or plastic, vial contents, or liquid nitrogen. The cryoprotectant DMSO is an irritant and may be harmful if inhaled, swallowed, or absorbed through the skin. Freezing mediums containing DMSO should be disposed of in compliance with local regulations.
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