**5. Current trends in testis tissue cryopreservation**

Since the first reports of successful germ cell transplantation and xenografting of testis tissue raised new interest in this field, several promising cryopreservation protocols have been introduced. Perhaps not surprisingly, the results differed and at times conflicted depending on the tissue donor species/developmental stage. These first reports of cryopreservation of pig and mouse testis tissues were based on DMSO-based slow freezing protocols originally developed for isolated testis cells or for ovarian tissue, respectively (Honaramooz et al., 2002a; Schlatt et al., 2002). Later, other detailed studies comparing multiple protocols showed high cell viability with programmed slow-freezing of immature mouse testis tissue using 1.5M DMSO as a cryoprotectant (Milazzo et al., 2008; Traverse et al., 2011). DMSO has also been found to be a more suitable cryoprotective agent than ethylene glycol for immature mouse and rat testis tissue (Goossens et al., 2008; Jezek, 2001). Shinohara et al. (2002) reported the birth of mouse offspring from sperm retrieved from cryopreserved pre-pubertal testis tissue with DMSO after transplantation under tunica albuginea of the recipient testes (Shinohara et al., 2002). Similar results were obtained using primate testis tissue, where 1.4M (but not 0.7M) DMSO was able to protect some of the developmental potential of grafts from rhesus monkeys (Jahnukainen et al., 2007) but the 0.7M DMSO protocol was successful for cryopreservation of

Cryopreservation of Testicular Tissue 223

experimental conservation of gonadal tissue and cells by cryopreservation can serve as a

Many challenges are associated with the optimal maintenance of tissue structure and the subsequent functional restoration of cryopreserved samples. It is intuitively known that optimal cryopreservation requires refinement of freezing and thawing rates, osmotic conditions, choice and concentration of cryoprotectants, and equilibration times in cryoprotective solutions. Indeed, improvement of all aspects of freezing techniques will ensure survival rates of tissue structure and subsequent functional restoration of cryopreserved cells within those tissues. Several studies have examined cryopreservation of testis cell suspensions or tissue fragments using glycerol, ethylene glycol, DMSO, or propanediol. In most cases, analyses of the cryopreserved samples lacked functional assessments of the preserved testicular cells/tissues. We now know that even if many cells of a multicellular system survive freezing and thawing, preservation of all functional compartments of the tissue is not guaranteed. Merely maintaining the physical characteristics of the cryopreserved testis tissue is not adequate, and an efficient approach to overcome the deficiencies in developmental

The author would like to thank the Natural Sciences and Engineering Research Council (NSERC) of Canada, and the Saskatchewan Health Research Foundation for grants to

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Abrishami M. (2009) Cryopreservation and xenografting of testis tissue. MSc Thesis.

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**7. Acknowledgement** 

Saskatoon.

324-31.

**8. References** 

human testis tissue (Wyns et al., 2007) at one age/developmental stage but not others (Wyns et al., 2008; Keros et al., 2005, 2007). Somewhat different from reports in other species, and after an extensive study of several strategies for cryopreservation of immature testis tissue, we concluded that glycerol was a better cryoprotectant for pig tissues (Abrishami et al., 2010a). These results suggest that each species and donor developmental age may need a different cryopreservation protocol, with a concomitant need to adjust the concentration of cryoprotectant or even adopt different cryoprotectants. These differences may be related to testicular architecture, morphology, or lipid composition.

In a first report of immature testis tissue vitrification, we also showed maintenance of cell viability and developmental potential to actively (re)establish complete spermatogenesis after xenografting into immunodeficient mice (Abrishami et al., 2010a). Recently, similar or much higher cell viability results were obtained using immature mouse testis tissue with vitrification compared with conventional slow freezing (Gouk et al., 2011; Curaba et al., 2011). With proper tissue handling, and the use of an appropriate choice of final cryoprotectant exposure, vitrification can provide preferential conditions for tissue freezing with proven superior results in restoration of immature testis tissue. Vitrification also does not require the extensive laboratory equipment commonly used for programmed slow freezing; however, direct plunging of tissues into liquid nitrogen, a common procedure in routine vitrification, poses a greater risk of contamination. The solid-surface vitrification of testis tissue (**Fig. 3**) is an easy, safe, and applicable cryopreservation technique for the preservation of tissue structural integrity and developmental potential.
