**8. References**

94 Current Frontiers in Cryobiology

prepubertal boys who are not capable of producing mature sperm. Testicular tissue cryopreservation has been reported in boys with cryptorchidism to preserve fertility (Bahadur et al., 2000). Cryopreserved testicular tissues can be autografted to restore reproductive functions; however recurrence of neoplastic process is a concern in oncology patients and such procedures are still considered to be experimental (Hwang & Lamb, 2010). A multi-disciplinary team approach is important to ensure that patients have the

The posthumous use of semen is an entirely separate and complex ethico-legal subject. The ethical and legal aspects of posthumous assisted reproduction have been recently addressed by the European Society of Human Reproduction and Embryology Task Force on Ethics and

Human spermatozoa can be successfully cryopreserved and utilized. Cryopreservation now

patients suffering from degenerative illnesses such as diabetes or multiple sclerosis;

Normozoospermic semen samples appear to be more tolerant to damage induced by freezing and thawing compared with oligozoospermic or asthenozoospermic samples. Cryopreservation of surgically retrieved epididymal and testicular spermatozoa is challenging, but a valuable component in effective treatment and management of severe male factor infertility. Cryopreservation of low numbers or single spermatozoa has multiple biological and technical aspects yet to be worked out; therefore, further research is required to introduce this technique into clinical practice. During cryopreservation, cells and tissue undergo dramatic transformation in chemical and physical characteristics as temperature drops from +37 to -196°C, thus risking cryoinjury. Velocity of cooling and warming is crucial and inaccurate cooling or thawing rates negatively correlate with sperm survival.

Spermatozoa cannot survive slow freezing without CPAs; CPAs have to be used at low concentrations with minimum exposure as CPAs are toxic and can cause osmotic damage. Gradual, stepwise introduction before freezing and removal of CPAs after thawing is essential. Conventional slow freezing with CPAs can offer cooling rates of 1–10°C/min. Vitrification, currently only an experimental technique, allows for extremely rapid freezing at rates of up to a 1000°C/min. LN2 can offer long-term survival of spermatozoa due to essentially absent metabolic activity and aging of cells and tissues in the frozen state. Rigorous standards of operation and quality control are essential for sperm banks. Social, psychological, legal and ethical issues surrounding sperm banking are very complex and

The vitrification method uses no specially developed cooling program; it does not need permeable cryoprotectants; it is much faster, simpler and cheaper; and it can also provide a

plays an essential role in fertility preservation under the following scenarios:

 cancer patients undergoing gonadotoxic chemotherapy or radiation. patients undergoing certain types of pelvic or testicular surgeries

 men undergoing surgical sterilization such as vasectomy screening and quarantine of donor semen samples

opportunity to preserve their fertility potential if they elect to do so.

Law (ESHRE, 2006).

**7. Conclusions** 

couples undergoing infertility treatment.

spinal cord disease or injury.

must be considered in each case.


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**4** 

*1USA 2China* 

**Prevention of Lethal Osmotic Injury to** 

**Cells During Addition and Removal of** 

*2University of Electronic Science and Technology of China, Chengdu,* 

Dayong Gao1 and Xiaoming Zhou2 *1University of Washington, Seattle, WA* 

**Cryoprotective Agents: Theory and Technology** 

Significant survival of cryopreserved cells became a reality only after the discovery and the use of cell-membrane-permeating cryoprotective agents (CPAs) (e.g. glycerol, Polge et al, 1949). Before freezing, one or various CPAs should be added to cell suspensions to prevent the cells from the cryoinjury during the freezing and thawing processes. Unfortunately, the CPAs, themselves, may have chemical toxicity to cells after thawing at room temperatures (Katkov el al, 1998). Therefore, a post-thaw washing of CPAs is required to remove CPAs from cells prior to scientific or medical applications. However, the addition of CPAs to cells before freezing and the removal of CPAs from cells after thawing may cause serious cell loss

"One-step" methods were formerly used for addition/removal CPAs. During the "onestep" CPA addition process, cells are directly (one-step) placed in a solution that is hyperosmotic with respect to the permeating CPA but isosmotic with respect to the impermeable salts/electrolytes. Cells first shrink because of the osmotic efflux of intracellular water and then increase in volume as the CPA permeates and as water concomitantly reenters the cells (as shown in Figure 1a). During the "one-step" CPA removal process, cells with a high intracellular concentration of CPA are directly exposed to an isotonic salt solution without CPA. Cells will swell because of an osmotic influx of extracellular water and then decrease in volume as the CPA diffuses out of the cells and as water concomitantly moves out (as shown in Figure 1b). As a result of these two aspects (i.e. addition and removal of CPAs) of the cryopreservation procedures, the cells may experience severe osmotic volume excursion causing significant cell "osmotic" injury (Sherman, 1973; Mazur and Schneider, 1984, 1986; Penninckx et al, 1984; Leibo, 1986, Crister et al, 1988a,

Several possible reasons for the osmotic injury have been proposed, including (i) rupture of the cell membrane in hypo-osmotic conditions (i.e. expansion lysis); (ii) the water flux hypothesis: frictional force between water and potential membrane 'pores' caused cell membrane damage (Muldrew and McGann, 1994); (iii) the minimum volume hypothesis:

and damage if the processes are not properly handled.

**1. Introduction** 

Meryman, 2007).

