**3.3 Protection mechanism and toxicity of cryoprotectants**

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

Cryopreservation of Testicular Tissue 215

Cryopreservation of cells within intact tissues is obviously more demanding than for cells within suspensions. Theoretical differences include heterogeneity of cells, slower rates of solute diffusion, and heat exchange through the mass of a complex tissue. However, judging from evidence from other tissue types, if a sufficient concentration of CPAs is provided, finding a proper cooling rate can yield high survival for different cell types within the tissue (Pegg, 2007). Critical factors for effective cryopreservation, such as cell permeability to water or CPA and subsequent osmotic changes, are directly affected by the rate of cooling (Mazur, 1990). Therefore, finding the optimal cryopreservation protocol for testicular tissue of a particular species/maturational state depends on the application of a proper concentration

Slow (controlled) freezing is considered the conventional method for cryopreservation of testicular tissue, in which the CPA is used at low concentrations (usually 0.5 to 2 M) to minimize both cell damage and CPA toxicity. During slow freezing (*e.g.,* -1°C/min), the CPA is given a chance to slow down the formation of extracellular ice crystals (and prevent the intracellular ones) but especially to moderate the indirect solution effects as freezing proceeds. However, prolonged exposure to CPA before completion of cryopreservation can also cause cell toxicity (Fuller, 2004). On the other hand, if the cell is cooled more rapidly, then water will not leave the cells fast enough to avoid intracellular freezing, which is very damaging to the cells (Pegg, 2007). Using automated systems, freezing curves (**Fig. 2**) can be

**A B**

customized to maximize cell viability after cryopreservation of the tissue.

Fig. 2. A programmable automated freezing system. Although requiring larger capital investments, automated cell/tissue freezing systems (**A**), consisting of a freezing chamber attached to a computer and a liquid nitrogen tank, allow customization of the freezing curve (**B**) to achieve pre-defined temperatures (Y-axis) for desired lengths of time (X-axis), in an

As indicated earlier, the formation of extracellular ice, which may not pose a problem for freezing of cell suspensions, is likely the main problem for tissues. Therefore, an alternative route to avoid ice crystal formation and solute damage within the tissue is to avoid ice crystal formation altogether using transformation of aqueous milieu of the cell/tissue to the amorphous character of a glassy state, known as vitrification. Vitrification is a cryopreservation method in which ice crystal formation is prevented because the cells or tissues are exposed to very high concentrations of CPAs (*e.g.,* 5 to 8 M) and undergo ultra rapid freezing rates (*e.g.,* up to -2500°C/min) (Fuller, 2004; Pegg, 2002, 2007). However, this

of the cryoprotectant with a suitable cooling rate.

accurate and consistent manner.

Cryoprotective agents are known to act through different pathways to protect cells against freezing injuries. This includes modulation of hydrogen bonding and interaction with water molecules, which give CPAs solubility and high permeability across cell membranes (Fuller, 2004). As a second mechanism, CPAs may provide a salt-buffering effect. During freezing, cells experience osmotic dehydration and shrinkage; therefore, the addition of CPAs into the cells maintains salt dilution. Basically, the CPA replaces water in cells, which dilutes the intracellular salts and prevents intracellular crystal formation. The amount of CPAs and water that permeates into the cells depends on the concentration of permeable solutes and the final cell volume. The properties of CPAs and those of cell membranes will influence the degree of cryoprotection for different cell types (Fuller, 2004; Fuller & Paynter, 2004). A third potential pathway is the stabilization of biomembrane critical macromolecules. Under normal conditions, water stabilizes the membrane bilayers. Loss of water during cryopreservation may disrupt normal membrane permeability and damage the membrane itself. The CPAs stabilize proteins as well as phospholipid bilayers of cell membranes and help to protect the membrane against freezing and dehydration stresses (Crowe, et al., 1990). Studies have collectively demonstrated that CPAs, including DMSO and disaccharide sugars such as sucrose and trehalose, may electrostatically interact with membrane phospholipids to provide stabilization (Anchordoguy et al., 1987; Rudolph & Crowe, 1985). The fourth mechanism by which CPAs protect the cells and tissue is through scavenging oxygen free radicals and preventing oxidative stress to the cells (Fuller, 2004). CPAs block the action of unstable intermediate products, such as oxygen free radicals, by binding their hydrogen atoms to them (Benson, 2004; Fleck et al., 2000). The fifth possible pathway for the protective effects of CPAs is the inhibition of nucleation, through which ice formation occurs in the media. During cooling, initial heterogeneous nucleation sites, such as small particles, change in shape and increase in size within media, eventually reaching a stage that forms ice crystals. Alternatively, induced nucleation could be beneficial to provide consistent extracellular crystallization. This phenomenon is the basis for "seeding", which induces nucleation onto supercooled media enabling proper cryopreservation (Fuller, 2004). Seeding can be achieved by clamping the side of vials or straws with a forceps cooled in liquid nitrogen to stimulate local ice growth in the solutions. Intracellular nucleation can also be lethal or damaging for cells and tissues. Some CPAs, such as DMSO or glycerol, inhibit nucleation by increasing the high viscosity of intracellular water (Fuller, 2004). Nonpermeating CPAs, on the other hand, increase and promote cellular dehydration by increasing the extracellular solute concentration thereby reducing intracellular crystallization (Bagchi et al., 2008).

Despite the protective potential of CPAs, a side effect of their addition is cytotoxicity. Tissue tolerance to CPAs is limited and overexposure may cause damage (Pegg, 2002); however, measuring this toxicity is difficult to precisely assess (Fuller, 2004). Cytotoxicity is further exacerbated by increasing CPA concentrations during ice formation. Optimizing the freezing rate as well as the addition or removal of CPAs could reduce their toxicity (Pegg, 2002).

#### **3.4 Choice of cryopreservation strategies**

For cryopreservation of testicular tissue, two popular strategies are slow freezing and vitrification. These techniques differ mainly in the concentration of CPAs used.

Cryoprotective agents are known to act through different pathways to protect cells against freezing injuries. This includes modulation of hydrogen bonding and interaction with water molecules, which give CPAs solubility and high permeability across cell membranes (Fuller, 2004). As a second mechanism, CPAs may provide a salt-buffering effect. During freezing, cells experience osmotic dehydration and shrinkage; therefore, the addition of CPAs into the cells maintains salt dilution. Basically, the CPA replaces water in cells, which dilutes the intracellular salts and prevents intracellular crystal formation. The amount of CPAs and water that permeates into the cells depends on the concentration of permeable solutes and the final cell volume. The properties of CPAs and those of cell membranes will influence the degree of cryoprotection for different cell types (Fuller, 2004; Fuller & Paynter, 2004). A third potential pathway is the stabilization of biomembrane critical macromolecules. Under normal conditions, water stabilizes the membrane bilayers. Loss of water during cryopreservation may disrupt normal membrane permeability and damage the membrane itself. The CPAs stabilize proteins as well as phospholipid bilayers of cell membranes and help to protect the membrane against freezing and dehydration stresses (Crowe, et al., 1990). Studies have collectively demonstrated that CPAs, including DMSO and disaccharide sugars such as sucrose and trehalose, may electrostatically interact with membrane phospholipids to provide stabilization (Anchordoguy et al., 1987; Rudolph & Crowe, 1985). The fourth mechanism by which CPAs protect the cells and tissue is through scavenging oxygen free radicals and preventing oxidative stress to the cells (Fuller, 2004). CPAs block the action of unstable intermediate products, such as oxygen free radicals, by binding their hydrogen atoms to them (Benson, 2004; Fleck et al., 2000). The fifth possible pathway for the protective effects of CPAs is the inhibition of nucleation, through which ice formation occurs in the media. During cooling, initial heterogeneous nucleation sites, such as small particles, change in shape and increase in size within media, eventually reaching a stage that forms ice crystals. Alternatively, induced nucleation could be beneficial to provide consistent extracellular crystallization. This phenomenon is the basis for "seeding", which induces nucleation onto supercooled media enabling proper cryopreservation (Fuller, 2004). Seeding can be achieved by clamping the side of vials or straws with a forceps cooled in liquid nitrogen to stimulate local ice growth in the solutions. Intracellular nucleation can also be lethal or damaging for cells and tissues. Some CPAs, such as DMSO or glycerol, inhibit nucleation by increasing the high viscosity of intracellular water (Fuller, 2004). Nonpermeating CPAs, on the other hand, increase and promote cellular dehydration by increasing the extracellular solute concentration thereby reducing intracellular

Despite the protective potential of CPAs, a side effect of their addition is cytotoxicity. Tissue tolerance to CPAs is limited and overexposure may cause damage (Pegg, 2002); however, measuring this toxicity is difficult to precisely assess (Fuller, 2004). Cytotoxicity is further exacerbated by increasing CPA concentrations during ice formation. Optimizing the freezing rate as well as the addition or removal of CPAs could reduce their toxicity (Pegg,

For cryopreservation of testicular tissue, two popular strategies are slow freezing and

vitrification. These techniques differ mainly in the concentration of CPAs used.

crystallization (Bagchi et al., 2008).

**3.4 Choice of cryopreservation strategies** 

2002).

Cryopreservation of cells within intact tissues is obviously more demanding than for cells within suspensions. Theoretical differences include heterogeneity of cells, slower rates of solute diffusion, and heat exchange through the mass of a complex tissue. However, judging from evidence from other tissue types, if a sufficient concentration of CPAs is provided, finding a proper cooling rate can yield high survival for different cell types within the tissue (Pegg, 2007). Critical factors for effective cryopreservation, such as cell permeability to water or CPA and subsequent osmotic changes, are directly affected by the rate of cooling (Mazur, 1990). Therefore, finding the optimal cryopreservation protocol for testicular tissue of a particular species/maturational state depends on the application of a proper concentration of the cryoprotectant with a suitable cooling rate.

Slow (controlled) freezing is considered the conventional method for cryopreservation of testicular tissue, in which the CPA is used at low concentrations (usually 0.5 to 2 M) to minimize both cell damage and CPA toxicity. During slow freezing (*e.g.,* -1°C/min), the CPA is given a chance to slow down the formation of extracellular ice crystals (and prevent the intracellular ones) but especially to moderate the indirect solution effects as freezing proceeds. However, prolonged exposure to CPA before completion of cryopreservation can also cause cell toxicity (Fuller, 2004). On the other hand, if the cell is cooled more rapidly, then water will not leave the cells fast enough to avoid intracellular freezing, which is very damaging to the cells (Pegg, 2007). Using automated systems, freezing curves (**Fig. 2**) can be customized to maximize cell viability after cryopreservation of the tissue.

Fig. 2. A programmable automated freezing system. Although requiring larger capital investments, automated cell/tissue freezing systems (**A**), consisting of a freezing chamber attached to a computer and a liquid nitrogen tank, allow customization of the freezing curve (**B**) to achieve pre-defined temperatures (Y-axis) for desired lengths of time (X-axis), in an accurate and consistent manner.

As indicated earlier, the formation of extracellular ice, which may not pose a problem for freezing of cell suspensions, is likely the main problem for tissues. Therefore, an alternative route to avoid ice crystal formation and solute damage within the tissue is to avoid ice crystal formation altogether using transformation of aqueous milieu of the cell/tissue to the amorphous character of a glassy state, known as vitrification. Vitrification is a cryopreservation method in which ice crystal formation is prevented because the cells or tissues are exposed to very high concentrations of CPAs (*e.g.,* 5 to 8 M) and undergo ultra rapid freezing rates (*e.g.,* up to -2500°C/min) (Fuller, 2004; Pegg, 2002, 2007). However, this

Cryopreservation of Testicular Tissue 217

For successful cryopreservation of a complex vascularized tissue, such as testis tissue, the majority of essential cells need to be viable for the tissue to survive and retain its function. However, there is not yet a comprehensive and universally applied method for postthawing analysis of cryopreserved testis tissue; subsequently, multiple approaches have been used to assess tissue/cell viability and extent of cryogenic injuries. These approaches commonly include histopathological examination of tissue sections for morphological changes. Using light microscopy, for instance, such objective criteria as seminiferous cord/tubular diameter or cell density within tubule cross sections can be measured, or semiquantitative morphometric analyses applied to subjectively score such criteria as health or integrity of tissue compartments (Abrishami et al., 2010a; Curaba et al., 2011; Milazzo et al., 2008; Travers, et al., 2011). Transmitted electron microscopy, although not widely used, can be invaluable in the examination of subcellular components most likely to be affected by testis tissue cryopreservation, including cytoplasm integrity, nuclear membrane, and various organelles (Keros et al., 2007). Other valuable morphological analyses may include assessment of cell-specific changes, for example, using double-staining of proliferation markers (*e.g.,* Ki67) and MAGE-AH, vimentin, or CD34 for identification of spermatogonia,

Sertoli cells, or peritubular cells, respectively (Keros et al., 2007; Wyns, et al., 2007).

associated changes within the cryopreserved testis tissue (Milazzo et al., 2008).

A quantitative measure of tissue damage due to cytotoxicity after cryopreservation can be achieved through lactate dehydrogenase release assays (Curaba et al., 2011) or through viability assessment of dissociated cells after digestion of frozen-thawed tissues using Trypan blue exclusion assays or the various cell viability kits using a flow cytometer analyzer (Abrishami et al., 2010a; Gouk et al., 2011). Assessment of apoptosis, using for instance, caspase-3 (Wyns et al. 2008), or TUNEL assay for detection of DNA fragmentation provides insight into the extent of cell damage (Milazzo, et al., 2008). Detection of phophatidylserine translocation from the inner to the outer layer of the plasma membrane, using fluorescent-labelled Annexin V, also allows more targeted assessment of apoptotic-

Having merely high cell survival rates or lacking visible damage does not guarantee functional preservation of the tissue as a whole. A thorough post-thawing analysis should include a form of testing for the functionality of the cryopreserved tissue. Post-thawing *in vitro* organotypic culture of the cryopreserved testis tissue has allowed assessment of its survival in the shortterm (Curaba et al 2011; Keros et al., 2007) and measurement of its hormone release into culture media (Gouk et al., 2011). Perhaps more robust examination is provided by grafting, where the survival and developmental competence (both in terms of germ cell differentiation and androgen release) of the cryopreserved tissue *in vivo* as grafts allows a longer-term functional assessment (Abrishami et al., 2010a; Jahnukainen et al., 2007; Wyns et al., 2007).

To offer cryoprotection, the CPAs need to diffuse rapidly in and out of the tissue; therefore, the size of testis tissue samples undergoing cryopreservation can be an important intuitive consideration. The results of studies differ depending not only with respect to the donor species but also potentially on the protocols employed. For instance, while cryopreservation of immature rat testis using similar procedures demonstrated better results for 7.5 mg pieces than 15 mg pieces (Travers et al., 2011), cryopreservation of immature mouse testis using

**3.6 Post-thawing analysis** 

**3.7 Effects of tissue size** 

approach is compromised by the cytotoxic effects of CPAs at such high concentration, especially with increased exposure times (Fuller, 2004; Fuller & Paynter, 2004). For small volumes of cell suspension, CPA concentrations can be reduced somewhat by using very rapid cooling and warming rates. However, especially with increasing size and complexity of the tissue, the limits of temperature exchange rates are more restricted, hence the use of very high concentrations of CPAs are unavoidable (Pegg, 2007). To overcome this problem, the use of a combination of CPAs to improve vitrification while reducing toxicity has been suggested. Proper media may include disaccharides, such as sucrose or trehalose, and proteins or polymers (Kasai & Mukaida, 2004; Sutton, 1992). The optimal CPA concentrations and exposure times to prevent toxicity must be specifically considered for each tissue type. (Fuller & Paynter, 2004; Pegg, 2007). We have used a solid-surface vitrification method to minimize the volume surrounding the tissue pieces, while avoiding liquid nitrogen (LN2) vapour formation and preventing direct contact with LN2 to prevent potential contamination (**Fig. 3**, Abrishami et al., 2010a).

Fig. 3. Solid-surface vitrification procedure for testicular tissue fragments. After exposure of testis tissue fragments to differing concentrations of vitrification solutions for varying lengths of time (**A**), testis tissue fragments are placed on a sterile aluminum boat (**B**) floating on liquid nitrogen (**C**), then transferred into cooled cryovials (**D**) followed by plunging into liquid nitrogen (images modified from Abrishami, 2009).
