**3. Cryopreservation protocols**

Cryopreservation protocols are numerous and optimized for the cell type being frozen. These protocols fall into two major categories: equilibrium freezing and non-equilibrium freezing. Critical to either process is the partial elimination of water in the cell to avoid ice crystal damage. This chapter focuses on the two main methodologies employed in freezing reproductive cells.

Conventional slow freeze methodology is characterized as equilibrium freezing. Cells are preequilibrated in cryo-protecting agent (CPA) and gradual temperature depression in a controlled rate freezer optimized for the cell type being frozen is initiated. As super-cooling is achieved, a manual seeding process is required to initiate ice crystal formation outside of the cell. Continuous equilibrium of the cells is achieved by increasing the osmotic gradient initiated from the increasing proportion of ice in the surrounding medium. As a result, the cell dehydrates, thereby lowering the freezing point of the cell. At a point, with the cell being almost devoid of water, ice crystal formation is negligent and freezing occurs. This method is viewed as "forgiving" in practice, given increased pre-equilibration exposure times to relatively low concentrations of CPAs and as such promotes efficiencies by accommodating batch freezing of multiple samples.

discovery had led to successful semen storage of farm animals in 1953 and human sperm in 1964 [49]. Cryoprotectants are divided into two groups: intracellular (such as DMSO, glycerol and propylene glycol) and extracellular (such as sucrose, polyvinyl pyrrolidone, hydroxyethyl starch and dextran). One of their modes of action is lowering of the freezing point of the solution. Use of an intracellular cryoprotectant such as DMSO will prevent intracellular ice formation, while the seeding drives extracellular crystallization and the resulting increase in the osmolality of the cryosolution leading to cellular dehydration [50]. Cryoprotectants may also protect the cell membrane from the drastic changes occurring during the transition between fluid and solid states. Cryoprotectants may, however, be toxic to the cells, therefore over the years a relentless search for less toxic and efficient cryoprotectants ensued as well as for protocols combining several cryoprotectants in order to reduce individual solute concen-

In 1985, Rall and Fahy were able to successfully vitrify a strew of a relatively large volume (0.25 ml) containing mouse embryos with a mixture of DMSO, acetamide and polyethylene glycol that was snap frozen in liquid nitrogen [22]. Shortly after the publication on the first births from slow-frozen oocytes, the first pregnancy and live birth from vitrified oocytes was published [51]. Developments that led to this breakthrough included the understanding that the length of exposure of the cells to the vitrification solution should be minimized to reduce toxicity [52], as well as replacing DMSO with ethylene glycol and mixtures of several cryoprotectants [53]. These changes brought about successful vitrification of bovine, murine as well as human oocytes with multiple live births [54–56]. These advancements were accompanied by the development of appropriate carriers to facilitate rapid cooling such as open-pulled straws [57], electron microscopy grids [55] and nylon loops [58]. By the end of the 1990s, vitrification was applied to human embryos achieving live births with both blastocyst and cleavage stage embryos [59, 60]. The vitrification of oocytes, despite these developments, was lagging until the introduction of appropriate carriers. The development of Cryotop in Japan was the breakthrough that allowed the adoption of oocyte vitrification into routine clinical practice. It allowed for an extremely rapid cooling rate that was facilitated by a minimal volume and resulted in a very high survival rate and live births [61–63]. A few methodological modifications that were made to the kit simplified its use and supported its wide spread distribution. Two large comparative studies established its lead role in oocyte cryopreserva-

Cryopreservation protocols are numerous and optimized for the cell type being frozen. These protocols fall into two major categories: equilibrium freezing and non-equilibrium freezing. Critical to either process is the partial elimination of water in the cell to avoid ice crystal damage. This chapter focuses on the two main methodologies employed in freezing repro-

tration and the associated cell toxicity.

**2.5. The return of vitrification**

144 Cryopreservation in Eukaryotes

tion [64, 65].

ductive cells.

**3. Cryopreservation protocols**

As water excursion depends on the rate of cooling, risk can be mitigated. Rapid cooling can trap excess water inside the cell, leading to the formation of intracellular ice crystals, whereas slow cooling promotes high intracellular solute concentration by severe volume shrinkage. Both have deleterious effects on the cell.

In addition, cells that are cooled slowly are susceptible to cryo damage. Mechanisms of cryo damage include upregulation of heat and cold-shock proteins in response to cold temperatures [66, 67]. Induction of apoptosis [68], a mechanism of cryo damage, may not be immediately visible but delayed for several hours as cells try to recover from such cryopreservation stresses [69].

Largely contrasting this technology, non-equilibrium freezing was developed to overcome the many shortfalls of slow freeze methodology. Cells exposed to (usually 7.5–10%) lower strength cryoprotectant solution undergo dehydration and permeation with CPAs. Subsequent (30– 60 s) rapid exposure to higher (40%) hyperosmotic solution results in complete dehydration of the cell. The sample is plunged directly into liquid nitrogen. This avoids deleterious ice crystal formation with high concentrations of CPAs and supremely rapid cooling rates (15,000– 30,000°C/min). The extreme elevation in solution viscosity promotes solidification or a glasslike, suspended state as opposed to crystallization. This method requires high level manual dexterity, is labor-intensive, while offering decreased incubation times can consistently and reliably accommodate only one sample being frozen at a time. Highly skilled technicians may stagger multiple samples as per protocol, yet this leaves success rates subject to human variation. As a benefit, this method is easily introduced without the need of expensive equipment. Though unconventional, an added benefit is a comparable survival after repeat vitrification and warming of the same sample [70, 71].

Recent technological advancement into this freeze methodology is semi-automated vitrification. This platform allows simultaneous cryopreservation of up to four embryos in a closed system, addressing the long-term debate of cross-contamination in shared liquid nitrogen. Non-clinical preliminary data comparing GAVITM (Genea BIOMEDX) to commercial manual method in mouse and donated human blastocyst stage embryos is promising [72]. Further clinical evaluation and advancement to oocytes and all embryonic stages is under way. Given success of this platform, process standardization demonstrating improved ART efficiencies may implore the few labs resistant to convert to vitrification technologies to reconsider; albeit cost considerations excluded.
