**3.1 Prevent the ice formation**

The physical definition of vitrification is the glass-like solidification of solutions at low temperatures, without the formation of intracellular ice crystals. The vitrification technique is the solidification of a liquid without ice crystal formation. This phase is obtained by increasing the solute concentration of the vitrification media (increased viscosity that makes water solidify without expansion) or by using very fast cooling rate to avoid molecular rearrangement into ice crystals but into an amorphous glass. (Vajta, 2000). Consequently, the risk of injuries due to intra- or extracellular ice crystallization is avoided, which constitutes the main advantage of this technique.

Despite the fact that slow freezing is the most widely used cryopreservation technique, vitrification is a viable and promising alternative that is increasingly becoming more attractive to the commercial sector. Vitrification has been used for oocyte and tissue cryopreservation since the 1980s (Rall and Fahy, 1985). Since the first use of this approach in ovarian tissue cryopreservation, it has been well optimized, particularly by decreasing the concentration of CPAs used to reduce their toxicity, but also by increasing the cooling rate applied (Chen et al., 2006; Keros et al., 2009). These improvements of vitrification in ovarian tissue but also in embryo and oocyte vitrification place the vitrification as a gold standard method for germ cells cryopreservation (Saragusty and Arav, 2011). It has been suggested that with time, conventional slow freezing will be replaced entirely by vitrification techniques (Vajta and Kuwayama, 2006).

As mentioned above, DSC can be a precious aid for the study of vitrification. In fact, DSC allows the detection of phase transitions, both crystallization or glass transition, and can thus determine three thermal properties of vitrification solutions: the vitreous transition temperature, the critical cooling rate and the critical warming rate (Baudot et al., 2007). These three thermal properties are specific to each vitrification solution and could be used for a better utilization of the solutions. The critical cooling rate and the critical warming rate are the cooling and warming speeds above which the crystallization cannot occur between the vitreous transition temperature and the crystallization temperature. The calculation of these critical cooling and warming rates is based on a semi-empiric model developed by Boutron in 1986 according to the "classic" theory of crystallization (Boutron, 1986). This model reproduces well analytically the experimental results, but some approximations are introduced.

#### **3.2 Limit the ice formation**

226 Current Frontiers in Cryopreservation

Since recent years, several strategies are developed to avoid deleterious effects of the ice crystal formation during cooling, and thermodynamical approaches are more and more

The specimen and reference temperatures are controlled independently using separate (identical) ovens. The temperature difference between the sample and reference is maintained to zero by varying the power input to the two furnaces. This energy is then a measure of the

The physical definition of vitrification is the glass-like solidification of solutions at low temperatures, without the formation of intracellular ice crystals. The vitrification technique is the solidification of a liquid without ice crystal formation. This phase is obtained by increasing the solute concentration of the vitrification media (increased viscosity that makes water solidify without expansion) or by using very fast cooling rate to avoid molecular rearrangement into ice crystals but into an amorphous glass. (Vajta, 2000). Consequently, the risk of injuries due to intra- or extracellular ice crystallization is avoided, which constitutes

Despite the fact that slow freezing is the most widely used cryopreservation technique, vitrification is a viable and promising alternative that is increasingly becoming more attractive to the commercial sector. Vitrification has been used for oocyte and tissue cryopreservation since the 1980s (Rall and Fahy, 1985). Since the first use of this approach in ovarian tissue cryopreservation, it has been well optimized, particularly by decreasing the concentration of CPAs used to reduce their toxicity, but also by increasing the cooling rate applied (Chen et al., 2006; Keros et al., 2009). These improvements of vitrification in ovarian

enthalpy or hat capacity changes in the test specimen (relative to the reference).

associated to these strategies.

Fig. 7. Scheme of power-compensation DSC

**3.1 Prevent the ice formation** 

the main advantage of this technique.

As described in the introductive part, extracellular crystallization occurs during slow freezing. The penetrating CPAs like DMSO, PROH or ethylene glycol, are mainly used to reduce the volume of freezable ice within the cells, but their respective toxicity for the cells limit the potential concentration usable. However, since the last decades, nonpenetrating CPAs like disaccharides have been widely used in cryoprotective solution to improve cell preservation and reduce the penetrating CPA amount necessary. To our knowledge, the first use of sucrose as cryoprotectant of ovarian tissue was in 1996 with human ovaries (Hovatta et al., 1996). Sugars have been proved to play a role on ice crystal formation and stability (Kuleshova et al., 1999). Previous studies on mouse embryos revealed that sugars increase the homogeny ice transition temperature in freezing solutions. By this way, dehydration of the cells occurs when they are still permeable to water without reaching ice nucleation temperature. Moreover, the trehalose was reported to reduce the size of ice crystals and shorten their elongation during freezing (Sei et al., 2002).

On the basis of the contribution of DSC in other fields of cryopreservation [vitrification of ovary (Baudot et al., 2007), slow-freezing or vitrification of plants (Volk and Walters, 2006; Skyba et al., 2011), slow-freezing of aquatic crustaceans (Issartel et al., 2006)], our team chose a thermodynamic approach to study slow-freezing solutions for ovarian tissue. In fact, DSC allows the measure of the maximal quantity of ice formed in a solution (Qmax). Qmax corresponds to the definition of Boutron : "The heat of solidification are represented by the numbers q of grams of ice whose solidification at 0°C would liberate the same amount of heat as that from 100g of solution on crossing the corresponding peaks. They are close to the real quantities of ice crystallized in % (w/w) of the solution when it is ice which crystallizes. One obtains the heat in calories per 100g of solution by multiplying q by 79.78" (Boutron, 1984). Our team has thus quantified the quantity of ice formed in solutions used for the cryopreservation of ovarian tissue of different species (table 6).

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species 229

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19,


Table 6. Maximal quantity of ice crystallized (Qmax) in solutions used for the cryopreservation of ovarian tissue of bitch, doe rabbit and cow.

Then, our team decided to explore two research areas. On the one hand, DSC can compare the quantity of ice formed in two different cryopreservation solutions. Thus, it seems possible to select the most suitable cryopreservation solutions for slow freezing methods. The first results obtained in doe rabbit seem to confirm this hypothesis. In fact between two cryopreservation solutions tested, those for which the quantity of ice formed was the lowest, was also the one with the best biological results. On the other hand, for a given solution, DSC allows the measure of the quantity of ice formed for different freezing kinetics. Consequently, it seems also possible to select the most suitable kinetics according to the cryopreservation solutions.

#### **3.3 Promote the formation of a non-vulnerable extracellular ice**

Another approach to optimize cryopreservation process should be to control the ice crystal growth and shape in order to promote the less deleterious crystallization. It is already assumed that intracellular ice formation is lethal. Nonetheless, the recent observations of rabbit ovarian tissue by cryoscanning electronic microscopy and cryofracture reveal that depending on the cooling rate, the extracellular ice shape is modified. Moreover, according to these results the seeding temperature influences the shape and regularity of the ice crystals resulting in large uniform crystals when seeding was induced close to the solution melting point (Gosden et al., 2010).

Otherwise, a better understanding of intracellular ice formation can also be advantageous to improve cryopreservation processes. Indeed, Han *et al* (2009) investigated the size of intracellular ice crystals in mouse oocytes by cryomicroscopy. They conclude that increasing the concentration of macromolecules in the cells by increasing the extracellular non permeating solute concentration significantly reduced the required permeating CPA concentration for intracellular vitrification. Moreover they also observed that intracellular ice melting point was always lower than extracellular ice. Taken together, this information can be helpful to optimize the cryopreservation protocols.

Regarding DSC, a recent study showed that it is possible to differentiate the crystallization of intra and extracellular ice depending on freezing kinetics (Seki et al., 2009). Consequently, in addition to the measure of the quantity of crystallized ice, DSC can provide a better control of ice formation.
