**3.6 Post-thawing analysis**

216 Current Frontiers in Cryobiology

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

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

Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the CPA that has reached the internal compartments of a multicellular system must diffuse back through numerous membranes in the tissue, with each acting as a barrier. Therefore, optimal thawing and CPA removal procedures are also critical factors for cell/tissue survival after freezing (Bagchi et al., 2008). Earlier studies pointed out that consistent cooling and thawing rates (slow-freezing followed by slow-thawing, or fast-freezing followed by fast-thawing) can improve cell/tissue survival after cryopreservation (Whittingham et al., 1972). Moreover, extreme osmotic changes during CPA removal might damage the cells by extensive cell shrinkage or swelling associated with the rapid movement of water into the cell as compared to the slower movement of the CPA out of the cell. However, a limited amount of water replacement is needed to restore osmotic

contamination (**Fig. 3**, Abrishami et al., 2010a).

liquid nitrogen (images modified from Abrishami, 2009).

equilibrium and physiologic cell volume (Pegg, 2007).

**3.5 Thawing methods** 

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).

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

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).
