**3. Methodology for cryopreservation of testicular tissue**

A number of cryogenic strategies have been developed to serve as a means to maintain functional properties of the preserved cells and tissues. Apparently, the first successful cryopreservation of cells was carried out by accidental freezing of fowl sperm in diluents containing glycerol (Polge et al., 1949). Later, cryopreservation of bull sperm using glycerol (Polge & Lovelock, 1952; Smith, 1961), set the stage for revolutionizing the bovine artificial

Cryopreservation of Testicular Tissue 213

permeating the cells and protecting them against high solute concentration or ice crystal damage. Because various cryoprotectant agents (CPAs) permeate different cell types at varying rates, it is of benefit to understand the biophysics of cryopreservation to minimize

Two main rival theories have been proposed to explain cell damages due to freezing. One emphasizes the direct and primarily mechanical damage to live cells by ice crystals puncturing through the cell membranes, and the other highlights the secondary effects of ice formation via osmotic changes. Perhaps, both mechanisms are important and what is recently agreed upon is that for individual cells, for example those in suspensions, intracellular freezing is very hazardous, while the extracellular ice may not be as harmful (Pegg, 2007). Unlike cell suspensions, the cellular organization and structural composition of the tissue may be seriously affected by cryogenic damage through widespread extracellular ice formation (Hunt et al., 1982; Taylor & Pegg, 1983). Ice formation within a tissue, initiated in the extracellular space, leads to an osmotic gradient across the cell membranes, causing intracellular water to move toward the concentrated extracellular space surrounding the cells (Bagchi et al., 2008; Fuller, 2004). Due to the differential destructive effects of extracellular ice formation between cell suspensions and complex tissues, conventional approaches to cryopreservation of cells, even testis cells for instance, may not necessarily be suitable for multicellular tissues such as the testis tissue. Optimal cooling rates for various cell and tissue types have been shown to differ and be directly associated with the degree of water permeability of cell membranes at

different temperatures during freezing (Leibo et al., 1970; Mazur, 1990; Pegg, 2007).

consequences of ice crystal formation (Mazur, 2004).

**3.3 Protection mechanism and toxicity of cryoprotectants** 

shown to afford effective cryoprotection (Bagchi et al., 2008; Fuller, 2004).

When extracellular ice formation causes elevated solvent concentrations, it leads to cell dehydration; prolonged exposure to which can permanently damage cell membranes and destabilize proteins (Fuller, 2004). However, short exposure of cells to optimized concentrations of hypertonic media before freezing might protect them from retention of supercooled water within cells and subsequent crystallization during freezing (Fuller, 2004). When cooling is faster than optimal, intracellular ice formation could occur due to inadequate time for water to follow the osmotic gradient across the cell membrane (Fuller, 2004; Fuller & Paynter, 2004; Pegg, 2007). The osmotic tolerance of cells is another critical factor to be considered during addition and removal of different cryoprotectants. Physical destruction, subsequent organelle disruption, and functional damage are some of the known

Sufficient concentration of cryoprotectants could minimize ice crystallization and/or promote amorphous solidification (vitrification). Glycerol was introduced as a CPA in 1949 (Polge et al., 1949) and, a decade later, cryoprotective properties of dimethyl sulfoxide (DMSO) were also reported (Lovelock & Bishop, 1959). These two cryoprotectants have mainly been used since then as classic cryoprotective additives, although many other CPAs have been introduced. Permeating CPAs, such as DMSO, glycerol, methanol, propanediol, ethylene glycol, and dimethyl acetaldehyde, as well as non-permeating CPAs, including sucrose, dextran, albumin, polyvinyl pyrollidone, and hydroxyethyl starch, have also been

damage (Fuller & Paynter, 2004; Pegg, 2007).

**3.2 Freezing injuries** 

insemination industry. At about the same time, cryopreservation of unfertilized oocytes was also studied following exposure to glycerol and low temperatures (Smith, 1952). After initial success with *in vitro* embryo manipulation in the 1950s (McLaren & Biggers, 1958), research involving embryo freezing intensified. Many methods have now been developed for embryo cryopreservation and, since the 1980s, some have become routine procedures (Whittingham et al., 1972; Whittingham, 1977; Wilmut, 1972). Cryopreservation of mature oocytes has also been achieved (Fabbri et al., 2001; Porcu, 2001; Porcu et al., 1997), with high survival rates and development of normal pregnancies after *in vitro* fertilization (IVF).

Cryopreservation of structurally intact tissues in certain situations is more desirable than cryopreservation of isolated cells. This is especially important for complex tissues in which preservation of the target cells' functionality depends on that of other cell types present within the tissue. In case of testicular tissue, not only germ cells but also the intra-tubular supporting - Sertoli - cells as well as androgen producing interstitial - Leydig - cells are of particular interest. However, this requires devising suitable freezing protocols to maintain the existing relationship among different compartments of the tissue.

The first gonadal tissue to be successfully cryopreserved was ovarian tissue, using exposure to glycerol, resulting in preservation of cell viability and normal function after being autografted back into the animals (Deanesly, 1954; Green et al., 1956; Parkes, 1958). Subsequent reports of live rat offspring, sheep ovarian cyclic function, and pregnancy after grafting cryopreserved ovaries represented important steps in demonstrating the feasibility of this approach (Gosden et al., 1994; Parrot, 1960). Restoration of spermatogenesis was then obtained after cryopreserved testis cells were transplanted into recipient testes (Avarbock et al., 1996; Brinster & Nagano, 1998; Ogawa et al., 1999).

Cryopreservation of testicular tissue to be used as tissue *per se*, however, was not widely considered, perhaps due to lack of its potential applications. This need changed when we and others were first to show that cryopreservation of immature testis tissue prior to its xenografting can be done so as to maintain its potential for development of complete spermatogenesis (Honaramooz et al., 2002a; Schlatt et al., 2002). In a short period of time since then, major advances in cryopreservation of testicular tissue have opened new possibilities for preservation of male fertility in animals and humans. More recently, induction of complete spermatogenesis *in vitro* has further highlighted the importance of applying cryopreservation to testicular tissue for future applications. Overall, major advances have been made in the cryopreservation of reproductive tissues. The following sections review the primary contributing factors to be considered for optimal cryopreservation.

#### **3.1 Biophysics of cryopreservation**

A clear understanding of biophysical behaviour of cells at the time of freezing and exposure to different cryoprotectants is critical in providing conditions to improve the cell structural and functional potential after freezing-thawing. During slow rate of cooling, extracellular ice crystal formation begins with the presence of a nucleation site in the extracellular medium. Because ice is pure crystalline water, the extracellular space becomes hypertonic due to the removal of water as ice crystals develop. Intracellular water, therefore, moves outward across the cell membrane due to the differential osmotic gradient, and cells dehydrate and shrink. This is the opportunity when certain cryoprotective compounds come into play, permeating the cells and protecting them against high solute concentration or ice crystal damage. Because various cryoprotectant agents (CPAs) permeate different cell types at varying rates, it is of benefit to understand the biophysics of cryopreservation to minimize damage (Fuller & Paynter, 2004; Pegg, 2007).
