**6. Conclusion**

Although cryopreservation of isolated testis cells has been successfully achieved for animals and humans, only in the past 10 years has intense attention been paid to cryopreservation techniques aimed at maintaining the developmental potential of structurally intact testis tissue. Cryopreservation of testis tissue theoretically offers a practical method when other techniques such as cryopreservation of ejaculated sperm are not available or applicable. Preservation of testis tissue has many applications, including conservation of fertility for prepubertal boys undergoing gonadotoxic cancer therapies. Ovarian and testicular toxicity are the inevitable long-term consequences of certain therapeutic oncological regimens, leading to premature fertility failure or sterility in cancer patients. Cryopreservation of gonadal cells or tissue before high-dose gonadotoxic chemo- and radio-therapy may therefore be considered in a comprehensive treatment and recovery plan. This could provide an alternative method for preserving the fertility potential of prepubertal boys with cancer or azoospermic men, as spermatogenesis is not completed in these patients. Although successful gamete and gonadal tissue restoration could have major impact on the enhancement of fertility preservation, serious ethical implications associated with collection and preservation of human gametes and gonadal tissues have yet to be resolved. Salvaging the genetic potential of immature endangered and valuable animals through banking of gonadal tissue is also a subject of clinical significance in animal reproduction and conservation. Optimal cryoconservation methods could also be combined with transplantation, xenografting, or culturing techniques to overcome some of the complications in the biodiversity crisis of rare or endangered species. In fact, experimental methods for the generation of fertility-competent gametes from cryopreserved ovarian or testis tissues have paved the way for future clinical use in human patients. Therefore,

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

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

Although cryopreservation of isolated testis cells has been successfully achieved for animals and humans, only in the past 10 years has intense attention been paid to cryopreservation techniques aimed at maintaining the developmental potential of structurally intact testis tissue. Cryopreservation of testis tissue theoretically offers a practical method when other techniques such as cryopreservation of ejaculated sperm are not available or applicable. Preservation of testis tissue has many applications, including conservation of fertility for prepubertal boys undergoing gonadotoxic cancer therapies. Ovarian and testicular toxicity are the inevitable long-term consequences of certain therapeutic oncological regimens, leading to premature fertility failure or sterility in cancer patients. Cryopreservation of gonadal cells or tissue before high-dose gonadotoxic chemo- and radio-therapy may therefore be considered in a comprehensive treatment and recovery plan. This could provide an alternative method for preserving the fertility potential of prepubertal boys with cancer or azoospermic men, as spermatogenesis is not completed in these patients. Although successful gamete and gonadal tissue restoration could have major impact on the enhancement of fertility preservation, serious ethical implications associated with collection and preservation of human gametes and gonadal tissues have yet to be resolved. Salvaging the genetic potential of immature endangered and valuable animals through banking of gonadal tissue is also a subject of clinical significance in animal reproduction and conservation. Optimal cryoconservation methods could also be combined with transplantation, xenografting, or culturing techniques to overcome some of the complications in the biodiversity crisis of rare or endangered species. In fact, experimental methods for the generation of fertility-competent gametes from cryopreserved ovarian or testis tissues have paved the way for future clinical use in human patients. Therefore,

testicular architecture, morphology, or lipid composition.

preservation of tissue structural integrity and developmental potential.

**6. Conclusion** 

experimental conservation of gonadal tissue and cells by cryopreservation can serve as a platform for further evaluation of the potential for long-term storage.

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 (re)establishment of spermatogenesis is also required.
