**3.7 Effects of tissue size**

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

Cryopreservation of Testicular Tissue 219

using human testes (Schlatt et al., 1999; Brook et al., 2001). Although autologous/homologous transplantation of germ cells for humans is currently considered purely experimental, one possibility for prepubertal human testis samples taken and frozen prior to treatments is to isolate testis cells and transfer them back to the individual. As a major problem with this approach is the risk of reseeding a systemic cancer, solutions to this (e.g., soring out tumour

We have expanded the technique for germ cell transplantation into farm animals (**Fig. 4**), showed the feasibility of SSC engraftment in unrelated recipient individuals (of the same species) without a need for immune-suppression, and further demonstrated the applicability of the approach through donor-derived sperm production by the recipients and birth of progeny carrying the donor characteristics (Honaramooz et al., 2002b 2003a, 2003b; Honaramooz & Yang, 2011). Therefore, although experimental at this stage, the approach may offer promise in salvaging genetic material from cryopreserved testicular

**C**

**D**

Fig. 4. Schematic overview of germ cell transplantation from a donor male into the testes of a recipient. The testes are collected from a donor animal (**A**), which could theoretically include post-mortem testis recovery from a recently deceased juvenile individual of an endangered species. The testis tissue could be cryopreserved (**B**) until conditions for its use are in place. At the time of transplantation, a single-cell suspension is prepared and the cells are infused into the seminiferous tubules of a recipient animal (**C**). Mating of the recipient (**D**) produces progeny (**E**), some of which will carry the donor genome (image modified

**E**

**B**

Another potential strategy for the use of cryopreserved testis tissue is represented by testis tissue xenografting. Grafting of both fresh and cryopreserved testis tissue fragments from

cells) and other safety issues are under investigation.

tissue from immature endangered species.

**A**

from Honaramooz et al., 2003b).

**4.2 Testis tissue (xeno)grafting** 

whole testes with punctured tunica albuginea was deemed more suitable than using whole testes with intact tunica, whole testes without tunica, or testis halves (Gouk et al., 2011). Mouse testes have considerably less connective tissue content than most other species; therefore, tissue fragment size is especially a concern for testis tissues from species with higher interstitial tissue density. For cryopreservation of (cryptorchid) testes from prepubertal boys, fragments sizes of 2-9 mm3 were used successfully (Wyns et al., 2007). We also reported that immature porcine testis tissues undergoing the same cryopreservation treatments were not affected by the original size of the testis tissue fragment (5, 15, 20, or 30 mg) (Abrishami et al., 2010a). Although not used for cryopreservation, no effect of tissue sample size was observed for one-wk old piglet testes (as intact or fragments of 100 or 30 mg) when used for hypothermic preservation for 6 days (Yang et al., 2010). It remains to be seen if whole human testes can be cryopreserved as has been accomplished for whole ovaries (Courbiere et al., 2006; Jadoul et al., 2007; Martinez-Madrid et al., 2007).
