**Theoretical evaluation of two-step glycerol removal using an osmotic buffer**

A two-step removal of cryoprotective agent from human spermatozoa using a nonpermeating solute as an osmotic buffer has been previously used to avoid osmotic injury in other cell types (Rowe et al., 1968; Leibo and Mazur, 1978; Watson, 1979). The steps involved in this approach are (i) the cryoprotective agent is directly removed and cell swelling is reduced by transferring cells with the cryoprotective agent to a hyperosmotic medium (osmotic buffer) of non-permeating solutes; and (ii) the cells in the osmotic buffer are rehydrated by directly transferring them to isotonic solution. Since current results showed that 600 mOsmol was the hyperosmotic upper tolerance limit for human spermatozoa to maintain 95% motility, the osmolality of the osmotic buffer medium should not exceed 600

Prevention of Lethal Osmotic Injury to Cells

condition (286 mOsmol) in one step.

Castino et al,1996; Arnaud et al 2003).

glycerol from human red blood cells (RBCs)

During Addition and Removal of Cryoprotective Agents: Theory and Technology 121

removal of glycerol (Table 3) using sucrose as an osmotic buffer was 43±5.3% ( *X* SEM , n=15). The experimental result agreed well with the predictions generated from the computer simulations. Data analyses indicated that the different glycerol removal procedures caused different motility losses (P<0.001 between any two procedures). Over 90% of spermatozoa maintained membrane integrity under all experimental conditions.

Fig. 15. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was removed from spermatozoa by two steps using a 'hyperosmotic buffer' solution. Step 1: 1.0 M glycerol was removed from spermatozoa by one -step exposure of spermatozoa to 600 mOsmol hyperosmotic (salt+sucrose) solution without glycerol. Step 2: Spermatozoa in the 600 mOsmol solution were returned to isotonic

Example 2: Development of a novel dilution-filtration method and instrument to remove

Cryopreservation has been widely used today around the world for long term preservation of RBCs. In the USA, the FDA has approved the storage of frozen RBCs at -80°C for as long as 10 years (Meryman, 2007). However, the glycerol in RBCs must be reduced to final concentration below 1% before infusion to prevent hemolysis (Valeri et al, 2001). The step of removing CPAs may cause serious cell loss due to the cell volume excursion induced by osmotic disequilibria (Meryman, 2007). In the past decades, many efforts have been made to improve the process (Rowe et al, 1968; Meryman et al, 1972, 1977; Valeri et al, 1975, 2001;

Currently, multi-step centrifuging methods are most commonly used, and some of them can achieve favorable results (Rowe et al, 1968; Meryman et al, 1972, 1977; Valeri et al, 1975, 2001). However, the procedures are very difficult and time consuming for manual operation due to the large cell suspension volume or high CPA concentration. In addition, most of the systems are not closed and are thus open to contamination (Castino et al,1996; Valeri et al, 2006). Automatic centrifuging systems may significantly reduce human labor and

mOsmol. Using this liming criterion, a hyperosmolality of 600 mOsmol would be expected to provide the maximum 'buffer effect' to reduce sperm volume swelling during the first step of glycerol removal. Sperm volume excursion during this two-step glycerol removal process was calculated and is shown in Figure 15. It was predicted that the maximum volume spermatozoa would achieve is 1.25 times (15%) the isotonic cell volume, which is higher than the UVL of human spermatozoa, and could be expected to cause >40% sperm motility loss, as predicted from Figure 10.

Fig. 14. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was removed from spermatozoa by four, six and eight fixed-molarity steps. The dotted lines in this figure indicate the upper volume limit, 1.1, below which >95% of spermatozoa can maintain the motility. The four- or six-step dilution results in a cell volume excursion causing >5% motility loss.

#### **Results from experimental examination**

Glycerol was added to or removed from human spermatozoa using stepwise procedure to test the theoretical predictions. A one-step addition resulted in ~19.2% sperm motility loss or 81.8±8.7% ( *X* SEM , n=15) motility recovery, while the four-step FMS or FVS addition significantly (P<0.001) increased in the motility recovery to 93.5±5.6% ( *X* SEM , n=15) or 91±4.8% ( *X* SEM , n=15) respectively. During different glycerol removal procedures (c.f. Table 2), <30% (28.5±3.8%, n=15) of motile spermatozoa kept their motility after a one-step removal of 1.0 M glycerol, while the majority of spermatozoa (92±8.2%, n=15) maintained motility after the eight-step FMS removal. In comparison, only 62±5.8% of spermatozoa maintained motility after eight-step FVS removal. The motility recovery after a two-step

mOsmol. Using this liming criterion, a hyperosmolality of 600 mOsmol would be expected to provide the maximum 'buffer effect' to reduce sperm volume swelling during the first step of glycerol removal. Sperm volume excursion during this two-step glycerol removal process was calculated and is shown in Figure 15. It was predicted that the maximum volume spermatozoa would achieve is 1.25 times (15%) the isotonic cell volume, which is higher than the UVL of human spermatozoa, and could be expected to cause >40% sperm

Fig. 14. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was removed from spermatozoa by four, six and eight fixed-molarity steps. The dotted lines in this figure indicate the upper volume limit, 1.1, below which >95% of spermatozoa can maintain the motility. The four- or six-step dilution

Glycerol was added to or removed from human spermatozoa using stepwise procedure to test the theoretical predictions. A one-step addition resulted in ~19.2% sperm motility loss or 81.8±8.7% ( *X* SEM , n=15) motility recovery, while the four-step FMS or FVS addition significantly (P<0.001) increased in the motility recovery to 93.5±5.6% ( *X* SEM , n=15) or 91±4.8% ( *X* SEM , n=15) respectively. During different glycerol removal procedures (c.f. Table 2), <30% (28.5±3.8%, n=15) of motile spermatozoa kept their motility after a one-step removal of 1.0 M glycerol, while the majority of spermatozoa (92±8.2%, n=15) maintained motility after the eight-step FMS removal. In comparison, only 62±5.8% of spermatozoa maintained motility after eight-step FVS removal. The motility recovery after a two-step

results in a cell volume excursion causing >5% motility loss.

**Results from experimental examination** 

motility loss, as predicted from Figure 10.

removal of glycerol (Table 3) using sucrose as an osmotic buffer was 43±5.3% ( *X* SEM , n=15). The experimental result agreed well with the predictions generated from the computer simulations. Data analyses indicated that the different glycerol removal procedures caused different motility losses (P<0.001 between any two procedures). Over 90% of spermatozoa maintained membrane integrity under all experimental conditions.

Fig. 15. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was removed from spermatozoa by two steps using a 'hyperosmotic buffer' solution. Step 1: 1.0 M glycerol was removed from spermatozoa by one -step exposure of spermatozoa to 600 mOsmol hyperosmotic (salt+sucrose) solution without glycerol. Step 2: Spermatozoa in the 600 mOsmol solution were returned to isotonic condition (286 mOsmol) in one step.

Example 2: Development of a novel dilution-filtration method and instrument to remove glycerol from human red blood cells (RBCs)

Cryopreservation has been widely used today around the world for long term preservation of RBCs. In the USA, the FDA has approved the storage of frozen RBCs at -80°C for as long as 10 years (Meryman, 2007). However, the glycerol in RBCs must be reduced to final concentration below 1% before infusion to prevent hemolysis (Valeri et al, 2001). The step of removing CPAs may cause serious cell loss due to the cell volume excursion induced by osmotic disequilibria (Meryman, 2007). In the past decades, many efforts have been made to improve the process (Rowe et al, 1968; Meryman et al, 1972, 1977; Valeri et al, 1975, 2001; Castino et al,1996; Arnaud et al 2003).

Currently, multi-step centrifuging methods are most commonly used, and some of them can achieve favorable results (Rowe et al, 1968; Meryman et al, 1972, 1977; Valeri et al, 1975, 2001). However, the procedures are very difficult and time consuming for manual operation due to the large cell suspension volume or high CPA concentration. In addition, most of the systems are not closed and are thus open to contamination (Castino et al,1996; Valeri et al, 2006). Automatic centrifuging systems may significantly reduce human labor and

Prevention of Lethal Osmotic Injury to Cells

**Theory of optimal operation protocol** 

procedure are described below.

**Basic Assumptions and Formulation** 

convection factors can be neglected.

derived by focusing on the extracellular solution:

respectively.

Source/Sink terms

During Addition and Removal of Cryoprotective Agents: Theory and Technology 123

Optimal operation protocol is defined here as the processes that minimize the operation time (to a final CPA concentration below 10g/L) as well as the osmotic cell volume excursion. A theoretical model was developed to predict the optimal operation protocols under the given experimental conditions (initial CPA concentration, cell density and total volume of cell suspension) and practical constraints. The detailed considerations for this

The theoretical model of the dilution-filtration system is developed (as shown in Fig.17) under the following assumptions: (1) Both intra- and extra-cellular solutions in cell suspension consist of water, a permeable CPA (e.g. glycerol) and an impermeable salt (e.g. NaCl); (2) Blood bag, hollow fibers and their connecting tubing are filled with cell suspension, and cells are uniformly distributed in the suspension; (3) Extracellular solution is diluted/filtrated immediately and evenly at the diluting/filtrating point when cell suspension circulates in the system; (4) Suspension flow is one dimensional, and the

Fig. 17. Theoretical modeling of the system. A: the overall system, and B: a control volume.

Based on the assumptions, a governing equation about the mass transfer process can be

<sup>1</sup> ( ) *e e*

where, A refers to effective mass transfer area, D refers to diffusion coefficient, *ϕe* refers to extracellular solute concentration (in osmolality), and S is the mass source/sink term,

*t Ax x* 

The source/sink term can be derived by temporarily ignored the diffusion term:

*DA S*

(19)

 

contamination (Valeri et al, 2001), but the expensive cost limits their application in many areas. Recently, Dialysis was considered as an alternative method by some researchers (Castino et al,1996; Arnaud et al 2003; Ding et al 2007,2010). It can remove CPAs efficiently; however, due to the non-uniformity of distribution of hollow fibers, the mass transport in dialyzer is too complicated to be controlled, especially in the unsteady state. In addition, dialysis method is not efficient to remove large molecular substances (Daugirdas, et al, 2006), such as cell fragment and the released protein from broken cells. These factors limit the use of dialysis method in some applications.

In clinic, hemofiltration, which involves dilution and filtration to remove toxins from blood, has been proved to have better controllability as well as ability of removing large molecular substances than hemodialysis (Daugirdas, et al, 2006). By referencing to hemofiltration, a dilution-filtration system is developed recently for removing CPAs (Zhou et al, 2011). The closed system helps to avoid contamination to cells, and the continuous and automatic process could provide particular advantage in efficiency especially for large-scale samples. The related research work is introduced in the following.
