**1. Introduction**

100 Current Frontiers in Cryobiology

Schlegel, P.N. & Su, L.M. (1997). Physiological consequences of testicular sperm extraction.

Schlegel, P.N.; Palermo, G.D.; Goldstein, M.; Menendez, S.; Zaninovic, N.; Veeck, L.L. &

Schuster, T.G.; Keller, L.M.; Dunn, R.L.; Ohl, D.A. & Smith, G.D. (2003). Ultra-rapid freezing

Sereni, E.; Bonu, M.A.; Fava, L.; Sciajno, R.; Serrao, L.; Preti, S.; Distratis, V. & Borini, A.

technique. *Reproductive Biomedicine Online,* 16, pp. 89-95, ISSN 1472-6483 Shimada, K. & Asahina, E. (1975). Visualization of intracellular ice crystals formed in very rapidly frozen cells at -27 degree C. *Cryobiology,* 12(3), pp. 209-218, ISSN 0011-2240 Thaler-DeMers, D. (2001). Intimacy issues: sexuality, fertility, and relationships. *Seminars in* 

The American Society for Reproductive Medicine. (2004). Guidelines for sperm donation.

Tomlinson, M. & Sakkas, D. (2000). Is a review of standard procedures for cryopreservation

Tomlinson, M. (2005) Managing risk associated with cryopreservation. *Human Reproduction,*

Thomson, L.K.; Fleming, S.D.; Aitken, R.J.; De Iuliis, G.N.; Zieschang, J.A. & Clark, A.M.

Tournaye, H.; Goossens, E.; Verheyen, G.; Frederickx, V.; De Block, G.; Devroey, P. & Van

Tyler, J.P.; Kime, L.; Cooke, S. & Driscoll, G.L. (1996). Temperature change in cryo-

Verza, Jr. S.; Feijo, C.M. & Esteves, S.C. (2009). Resistance of human spermatozoa to

Walmsley, R.; Cohen, J.; Ferrara-Congedo, T.; Reing, A. & Garrisi J. (1998). The first births

egg zonae. *Human Reproduction,* 13 (Suppl. 4), pp. 61-70, ISSN 0268-1161 Woods, E.J.; Benson, J.D.; Agca, Y. & Critser, J.K. (2004). Fundamental cryobiology of reproductive cells and tissues. *Cryobiology,* 48, pp. 146-156, ISSN 0011-2240 World Health Organization (1999). *WHO laboratory manual for the examination of human semen* 

Rosenwaks, Z. (1997). Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. *Urology,* 49, pp. 435-440, ISSN 0090-4295 Schover, L.R.; Rybicki, L.A.; Martin, B.A. & Bringelsen, K.A. (1999). Having children after

cancer. A pilot survey of survivors' attitudes and experiences. *Cancer,* 86(4), pp.

of very low numbers of sperm using cryoloops. *Human Reproduction,* 18(14), pp.

(2008). Freezing spermatozoa obtained by testicular fine needle aspiration: a new

needed? Safe and effective cryopreservation—should sperm banks and fertility centres move toward storage in nitrogen vapour? *Human Reproduction,* 15(12), pp.

(2009). Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. *Human Reproduction* 24(9), pp.

Steirteghem, A. (2004). Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects. *Human Reproduction Update,* 10(6),

containers during short exposure to ambient temperatures. *Human Reproduction,*

cryoinjury in repeated cycles of thaw-refreezing. *International Brazilian Journal of* 

and ongoing pregnancies associated with sperm cryopreservation within evacuated

*and sperm-cervical mucus interaction,* pp. 169-179, 4th ed. Cambridge University

*Human Reproduction,* 12(8), pp. 1688-1692, ISSN 0268-1161

*Oncology Nursing,* 17(4), pp. 255-262, ISSN 0749-2081

*Fertility and Sterility,* 82, pp. S9–12, ISSN 0015-0282

697-709, ISSN 0008-543X

788-795, ISSN 0268-1161

2460-2463, ISSN 0268-1161

2061-2070, ISSN 1460-2350

pp. 525-532, ISSN 1355-4786

11(7), pp. 1510–1512, ISSN 0268-1161

*Urology,* 35, pp. 581-590, ISSN 1677-6119

Press, ISBN 978-924-154-778-9, Cambridge, UK

20, pp. 1751-1756, ISSN 0268-1161

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 and damage if the processes are not properly handled.

"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, Meryman, 2007).

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:

Prevention of Lethal Osmotic Injury to Cells

Fig. 2. Multi-step method for addition and removal of a CPA

introduced and discussed in this chapter.

model, considering solute-solvent interactions.

formulized as follows.

i. one-parameter model (Mazur et al, 1974, 1976),

During Addition and Removal of Cryoprotective Agents: Theory and Technology 103

In the past, attempts to develop procedures for the addition and removal of CPAs have been made based primarily on empirical approaches, i.e. for a given cell type, various temperatures, CPA types and concentrations, and number of procedures or steps for CPA addition and removal were empirically tested to find an acceptable procedure. Typical techniques includes (i) a multi-step addition and multi-step removal of permeating CPAs (Watson, 1979) and (ii) a multi-step addition and two-step removal (using a non-permeating solute as osmotic buffer) of CPAs (Rowe et al., 1968; Mazur and Leibo, 1977; Leibo 1981). New CPA addition-removal methods and automated devices have recently been developed based on fundamental cell membrane transport theory and engineering approaches (Gao, et al, 1995; Gilmore et al, 1997; Katkov, 1998; Myrthe, et al ,2004, Zhou, et al, 2011), which are

**2. Cell membrane transport models and mathematical formulatins** 

<sup>i</sup> dVw

and Π*i* are the extracellular and intracellular osmotic pressures.

To date, a number of formalisms exist for describing the cell membrane transport process. These include a one-parameter model, a two-parameter model, and a three-parameter

The one-parameter model utilizes the hydraulic permeability (*Lp*) of cell membrane as the only parameter to describe the water transport across cell membrane. The model can be

where, *<sup>i</sup> Vw* is the volume of intracellular water, *Ac* is the area of cell membrane surface, Π*<sup>e</sup>*

dt *L Ap ce i* (1)

cell shrinkage in hyper-osmotic condition is resisted by cytoskeleton components, and the resultant interaction between shrunken cell membrane and the cytoskeleton damages the cells (Meryman, 1970); (iv) the maximum cell surface hypothesis: the cell shrinkage induces irreversible membrane fusion/change, and hence the effective area of cell membrane is reduced; when returned to isotonic condition, the cells lyse before their normal volume is recovered (Steponkus and Wiest, 1979); and (v) the solute loading hypothesis: hyperosmotic stress causes a net leak/influx of non-permeating solutes; when cells are returned to isoosmotic conditions, they swell beyond their normal isotonic volume and lyse (Mazur et al., 1972).

Fig. 1. Cell volume excursion during addition and removal of CPAs

In order to minimize osmotic injury, many efforts have been made and several techniques have been proposed. Basically, people utilize so-called "multi-step methods" instead of "one-step method" for addition and removal of CPAs, and the resulting cell recovery rate can be significantly improved. During the multi-step CPA addition process, solution with high CPA concentration is added into a cell suspension step by step and the CPA concentration in the cell suspension increases slowly and gradually. During the multi-step CPA removal process, an isotonic salt solution is added into the cell suspension step by step, and then by means of centrifugation CPAs in the cell suspension are removed (Figure 2). Although to some extend multi-step method reduces osmotic damage of cells, it is complex to operate, requires more laboratory staffs, and costs more time, which makes the addition and removal procedures more expensive and difficult practically.

cell shrinkage in hyper-osmotic condition is resisted by cytoskeleton components, and the resultant interaction between shrunken cell membrane and the cytoskeleton damages the cells (Meryman, 1970); (iv) the maximum cell surface hypothesis: the cell shrinkage induces irreversible membrane fusion/change, and hence the effective area of cell membrane is reduced; when returned to isotonic condition, the cells lyse before their normal volume is recovered (Steponkus and Wiest, 1979); and (v) the solute loading hypothesis: hyperosmotic stress causes a net leak/influx of non-permeating solutes; when cells are returned to isoosmotic conditions, they swell beyond their normal isotonic volume and lyse (Mazur et al.,

 a b Fig. 1. Cell volume excursion during addition and removal of CPAs

and removal procedures more expensive and difficult practically.

In order to minimize osmotic injury, many efforts have been made and several techniques have been proposed. Basically, people utilize so-called "multi-step methods" instead of "one-step method" for addition and removal of CPAs, and the resulting cell recovery rate can be significantly improved. During the multi-step CPA addition process, solution with high CPA concentration is added into a cell suspension step by step and the CPA concentration in the cell suspension increases slowly and gradually. During the multi-step CPA removal process, an isotonic salt solution is added into the cell suspension step by step, and then by means of centrifugation CPAs in the cell suspension are removed (Figure 2). Although to some extend multi-step method reduces osmotic damage of cells, it is complex to operate, requires more laboratory staffs, and costs more time, which makes the addition

1972).

Fig. 2. Multi-step method for addition and removal of a CPA

In the past, attempts to develop procedures for the addition and removal of CPAs have been made based primarily on empirical approaches, i.e. for a given cell type, various temperatures, CPA types and concentrations, and number of procedures or steps for CPA addition and removal were empirically tested to find an acceptable procedure. Typical techniques includes (i) a multi-step addition and multi-step removal of permeating CPAs (Watson, 1979) and (ii) a multi-step addition and two-step removal (using a non-permeating solute as osmotic buffer) of CPAs (Rowe et al., 1968; Mazur and Leibo, 1977; Leibo 1981). New CPA addition-removal methods and automated devices have recently been developed based on fundamental cell membrane transport theory and engineering approaches (Gao, et al, 1995; Gilmore et al, 1997; Katkov, 1998; Myrthe, et al ,2004, Zhou, et al, 2011), which are introduced and discussed in this chapter.
