**2.5 Discussion**

An optimized method for addition and removal glycerol from cryopreserved human spermatozoa has been illustrated as an example. Although the mechanism(s) of the osmotic injury during cryopreservation is not clearly understood, the hypothesis has been tested and confirmed, i.e. human sperm volume excursion can be used as an indicator to predict possible osmotic injury to spermatozoa during glycerol addition and removal processes. Hence, the procedures used for testing the hypothesis provide a methodology to predict optimal protocols for cryoprotective agent addition/removal..

The FVS, multi-step procedure for the addition of glycerol to human spermatozoa before cryopreservation is a conventional, commonly used technique, i.e. 'drop by drop' (stepwise) addition of a solution with a relatively high glycerol concentration (the volume of each 'drop' is roughly constant) to the spermatozoa or sperm suspension in order to achieve a 0.6-1.0 M glycerol concentration in the final sperm suspension. In practice, the frozenthawed sperm samples containing glycerol are either washed for intrauterine insemination or four in-vitro fertilization or directly transferred into the lower female reproductive tract for artificial insemination (e.g. intercervical insemination). In both cases, the glycerol is abruptly removed from spermatozoa by direct exposure to near isotonic conditions. In the example, it was predicted by computer simulation, and confirmed experimentally, that a one-step removal of glycerol would cause a high frequency of sperm motility loss even without freezing. Based on the results, the FMS removal (≥8 steps) of 1.0 M glycerol is recommended. Within the scope of the present investigation, a four-step FMS addition of glycerol to spermatozoa to achieve a final 1.0 M glycerol concentration and an eight-step

221.8 30 93.64 2.60 204.1 27 85.92 3.39 229.8 29 91.64 12.26 219.0 23 90.91 4.90 217.6 25 91.38 3.39 210.0 28 90.76 6.56 216.7 24 81.60 3.80 200.5 29 94.18 7.34 204.5 21 92.24 5.35 205.0 30 93.80 6.08 **Mean** 212.9 27 91.19 5.57 **S.D.** 9.49 3 3.57 2.81

**Cell Count Recovery (%)** 

**Residual Glycerol (g/L)** 

**Thawed Blood Hct (%)** 

Table 6. *In-vitro* experiments of deglycerolization with dilution-filtration method

optimal protocols for cryoprotective agent addition/removal..

An optimized method for addition and removal glycerol from cryopreserved human spermatozoa has been illustrated as an example. Although the mechanism(s) of the osmotic injury during cryopreservation is not clearly understood, the hypothesis has been tested and confirmed, i.e. human sperm volume excursion can be used as an indicator to predict possible osmotic injury to spermatozoa during glycerol addition and removal processes. Hence, the procedures used for testing the hypothesis provide a methodology to predict

The FVS, multi-step procedure for the addition of glycerol to human spermatozoa before cryopreservation is a conventional, commonly used technique, i.e. 'drop by drop' (stepwise) addition of a solution with a relatively high glycerol concentration (the volume of each 'drop' is roughly constant) to the spermatozoa or sperm suspension in order to achieve a 0.6-1.0 M glycerol concentration in the final sperm suspension. In practice, the frozenthawed sperm samples containing glycerol are either washed for intrauterine insemination or four in-vitro fertilization or directly transferred into the lower female reproductive tract for artificial insemination (e.g. intercervical insemination). In both cases, the glycerol is abruptly removed from spermatozoa by direct exposure to near isotonic conditions. In the example, it was predicted by computer simulation, and confirmed experimentally, that a one-step removal of glycerol would cause a high frequency of sperm motility loss even without freezing. Based on the results, the FMS removal (≥8 steps) of 1.0 M glycerol is recommended. Within the scope of the present investigation, a four-step FMS addition of glycerol to spermatozoa to achieve a final 1.0 M glycerol concentration and an eight-step

**UNITS Thawed Blood** 

**2.5 Discussion** 

**Volume (ml)** 

FMS removal of 1.0 M glycerol from spermatozoa were predicted and shown to be acceptable procedures which minimize osmotic injury. From calculations, the minimum or maximum cell volumes after each step of FVS addition or removal were shown to be unequal, some of which may exceed the lower or upper volume limits of the cells. In contrast, from calculations, the minimum or maximum cell volumes after each step of FMS addition or removal of glycerol were shown to be relatively even (Figures 12 and 13). For a fixed number of steps, the minimum or maximum of cell volume excursion during glycerol addition or removal using the FMS approach is much smaller than that using the FVS approach (see Figures 12 and 13).

In the example, it was postulated that the sperm osmotic injury as a function of cell volume excursion must be determined to predict the optimal glycerol addition and removal procedures. However, the definition and determination of 'sperm injury' is dependent upon the assays used. In the example, sperm motility was used as a standard of sperm viability because of its relatively high sensitivity to osmotic changes and the requirement of sperm motility for functional viability. If sperm membrane integrity was chosen as the endpoint to evaluate the sperm viability, as shown in Figure 7, different osmotic tolerance limits would be obtained. One can readily repeat the same procedures to predict the extent to which spermolysis is caused by the different glycerol addition/removal procedures used in the example, based on the information provided in Figure 5. For example, it was found (Figure 7) that >85% of spermatozoa maintained membrane integrity when they were returned to isotonic condition after having been exposed to anisosmotic conditions ranging from 90 and 700 mOsmol. The corresponding sperm volume excursion range was 0.7-2.1 times the isotonic sperm volume (Figure 9). From Figures 12 and 13, it can be seen that a one-step addition and one-step removal of 1.0 M glycerol would result in a minimum relative sperm volume of 0.72 and maximum volume of 1.68 respectively, which did not exceed the sperm volume excursion range 90.7-2.1 times relative volume) for maintaining >85% sperm membrane integrity. Based on this information, one can predict that the majority (>85%) of spermatozoa would maintain membrane integrity even using one-step addition and one-step removal of glycerol.

A dilution-filtration system for removing CPAs from cryopreserved cell suspension was also introduced here. The system realized continuous processing of cell suspension and the dilution & filtration were conducted simultaneously, thus it can achieve much better efficiency than traditional multi-step centrifuging methods. Moreover, dilution in the system is conducted to cell suspension flow in tubing but not whole suspension in container, thus the mixing process should be much rapider and then the osmotic disequilibrium during dilution can be significantly reduced.

A theoretical model was established to simulate the specific process. Based on the model, cell volume excursion and the variation of CPA concentration during the dilutionfiltration process can be simulated. Theoretical analysis indicates the operation parameters, especially the flow rate of diluent, are critical for the dilution-filtration method. In the previous studies concerning removing CPAs with hollow fibers (Castino et al, 1996; Arnaud et al 2003; Ding et al, 2007, 2010 ), only the protocols with constant flow rates were discussed. However, it was found to be difficult to balance the requirements in removing efficiency and cell safety. This problem also exists in the presented dilution-

Prevention of Lethal Osmotic Injury to Cells

During Addition and Removal of Cryoprotective Agents: Theory and Technology 133

Curry, M.R. and Watson, P.F. (1994) Osmotic effects on ram and human sperm membranes

Daugirdas, J. T., Blake, P., Ing, T. S. and Blagg, C., 2006, Handbook of Dialysis, Fourth

Ding, W. P., Yu, J. P., Woods, E., Heimfeld, S., and Gao D. Y., 2007, "Simulation of removing

Ding, W. P., Zhou, X. M., Heimfeld, S., Reems, J., and Gao D. Y., 2010, "A Steady-State Mass

Du, Junying, Kleinhans, F.W., Mazur, P. and Crister, J.K. (1994) Human spermatozoa

Gao, D.Y., Mazur, P., Kleinhans, F.W., Watson, P.F., Noiles, E.E. and Crister, J.K. (1992)

Gao, D.Y., Ashworth, E., Watson, P.F., Kleinhans, F.W., Mazur, P. and Crister, J.K. (1993)

Gao, D.Y., Liu, J., Liu, C., McGann, L.E., Watson, P.F., Kleinhans, F.W., Mazur, P., Crister

Gilmore, J.A., Liu, J., Gao, D.Y. and Crister, J.K. (1997) Determination of optimal

Gilmore, J.A., McGann, L.E., Liu, J., Gao, D.Y., Peter, A.T., Kleinhans, F.W., and Crister, J.K.

Jequier, A. and Crich, J. (1986) Computer assisted semen analysis (CASA). In semen Analysis: A practical Guide. Blackwell Scientific, Boston, pp. 143-149. Jeyendran, R.S., Van der Ven, H.H., Perez-Pelaez, M., Crabo, B.G. and Zaneveld, L.J.D.

Katkov, I. I., 2000, "A Two-Parameter Model of Cell Membrane Permeability for Multisolute

Katkov, I.I., Katkova, N., Crister, J.K., and Mazur, P. (1998a) Mouse spermatozoa in high

chloride and sucrose on spermolysis. Biol. Reprod., 49, 112-123.

Modules," Journal of Biomechanical Engineering, 132(1), pp. 011002 Du, Junying, Kleinhans, F.W., Mazur, P. and Crister, J.K. (1993) Osmotic behavior of human

permeable cryoprotective agents from cryopreserved blood with hollow fiber

Transfer Model of Removing CPAs from Cryopreserved Blood with Hollow Fiber

glycerol permeability and activation energy determined by electron paramagnetic

Glycerol permeability of human spermatozoa and its activation energy.

Hyperosmotic tolerance of human spermatozoa: separate effect of glycerol, sodium

E.S. and Crister J.K. (1995). Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol, Human Reproduction 10, 1109-1122. Garner, D.L., Pinkel, D., Johnson, L.A. and Pace, M.M. (1986) Assessment of spermatozoal

function using dual fluorescent staining and flow cytometric analyses. Biol.

cryoprotectant and procedures for their addition and removal from human

(1995) Effect of cryoprotectant solutes on water permeability of human

(1984) Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod.

concentrations of glycerol: Chemical toxicity vs osmotic shock at normal and

Edition., Lippincott Willians & Wilkins, Philadelphia, pp.265-275.

modules," Journal of membrane science, 288(1), pp. 85-93.

spermatozoa studied by EPR. Cryo-letters, 14, 285-294.

resonance. Biochim. Biophys. Acta, 1994, 1-11.

spermatozoa. Human Reproduction 12, 112-118.

spermatozoa. Biol. Reprod. 53, 985-995.

Systems," Cryobiology, 40(1), pp. 64-83.

reduced oxygen concentration. Cryobiology 37, 325-338.

Cryobiology, 29, 657-667.

Reprod., 34, 127-138.

Fertil., 70, 219-228.

in relation to thawing injury. Cryobiology, 31, 39-46.

filtration method. Removing efficiency can be improved by using higher diluent flow rate, but the cell recovery rate may be seriously reduced in the way. Besides, when using a constant diluent flow rate, the profile of glycerol concentration is nearly exponential, i.e., the removing efficiency starts at the highest value but gradually decreases as the process going on. However, when using a stepwise increased diluent flow rate, the removing efficiency can be maintained at a high level for a quite long period. Moreover, theoretical analysis also indicates stepwise increasing of the diluent flow rate may not cause any extra cell damage. Therefore, a stepwise increased diluent flow rate is necessary to achieve both high cell recovery rates and efficient glycerol clearance when using the dilution-filtration system. In addition, it was also deduced by the theoretical analysis that the removing effect of an operation protocol is highly related to the initial volumes and cell densities of cell suspensions. Therefore, the optimal operation protocols should be specialized and various from case to case. The theoretical model provides an effective tool to find out the optimal protocols for given applications.

The system was also investigated experimentally with deglycerolization from cryopreserved blood, and the operation procedures were optimized based on the theoretical model. It is clearly indicated by the results that the dilution-filtration method is safe and efficient for deglycerolization from cryopreserved RBCs. Comparing to the automatic centrifuging method, the cell recovery rate and removing efficiency are similar, but the equipment cost of the dilution-filtration system is much lower and thus it can be applied in more areas. We can also believe that with properly selected operation parameters, this system can also be applied to various CPA removal applications. In addition, all the media are processed in a closed system, and thus the system should have further advantages in avoiding contamination. It is hopeful for the cells to have a long shelf life after washing. These suppositions will be verified by further experiments.

#### **3. References**


filtration method. Removing efficiency can be improved by using higher diluent flow rate, but the cell recovery rate may be seriously reduced in the way. Besides, when using a constant diluent flow rate, the profile of glycerol concentration is nearly exponential, i.e., the removing efficiency starts at the highest value but gradually decreases as the process going on. However, when using a stepwise increased diluent flow rate, the removing efficiency can be maintained at a high level for a quite long period. Moreover, theoretical analysis also indicates stepwise increasing of the diluent flow rate may not cause any extra cell damage. Therefore, a stepwise increased diluent flow rate is necessary to achieve both high cell recovery rates and efficient glycerol clearance when using the dilution-filtration system. In addition, it was also deduced by the theoretical analysis that the removing effect of an operation protocol is highly related to the initial volumes and cell densities of cell suspensions. Therefore, the optimal operation protocols should be specialized and various from case to case. The theoretical model provides an effective tool

The system was also investigated experimentally with deglycerolization from cryopreserved blood, and the operation procedures were optimized based on the theoretical model. It is clearly indicated by the results that the dilution-filtration method is safe and efficient for deglycerolization from cryopreserved RBCs. Comparing to the automatic centrifuging method, the cell recovery rate and removing efficiency are similar, but the equipment cost of the dilution-filtration system is much lower and thus it can be applied in more areas. We can also believe that with properly selected operation parameters, this system can also be applied to various CPA removal applications. In addition, all the media are processed in a closed system, and thus the system should have further advantages in avoiding contamination. It is hopeful for the cells to have a long shelf life after washing. These

Arnaud, F.G. and Pegg, D.E. (1990) Permeation of glycerol and propane-1,2-diol into human

Arnaud, F., Kapnik, E., and Meryman, H. T., 2003, "Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates," Platelets, 14(3), pp. 131-138. Bavister, B.D., Leibfriend, M.L. and Lieberman, G. (1983) Development of preimplantation

Brecher, M. E., 2002, Technical manual of the American Association of Blood Banks. 14th ed.,

Castino, F., and Wickramasinghe, S. R., 1996, "Washing frozen red blood cell concentrates using hollow fibers," Journal of membrane science, 110(2), pp. 169-180. Crister, J.K., Huse-Benda, A.R., Aaker, D.V., Arneson, B.W. and Ball G.D. (1988a)

Crister, J.K., Colvin, K.E. and Crister, E.S. (1988b) Effect of sperm concentration on computer

Society of Andrology. J. Androl., 9 (Suppl.), Abstr. 105, p.45.

embryos of the golden hamster in a defined culture medium. Biol. Reprod., 28, 235-

Cryopreservation of human spermatozoa, 3. The effect of cryoprotectants on

assisted semen analysis results. Abstracts of the 1988 Annual Meeting of American

to find out the optimal protocols for given applications.

suppositions will be verified by further experiments.

platelets. Cryobiology, 27, 107-118.

motility. Fertil. Steril., 50, 314-320.

American Association of Blood Banks, Bethesda.

**3. References** 

247


Prevention of Lethal Osmotic Injury to Cells

Biol. Reprod., 48, 99-109.

156.

21, 25-32.

19-128.

231-350.

218.

928-932.

freezing injury. Biophys. J., 66, 532-541.

range +25 °C to -10°C," Ph.D. thesis, MIT.

dehydration at law temperatures, Nature, 164, 666-676.

human semen banking. Fertil .Steril., 24, 397-412.

SAS Institute. Inc., Cary, NC. Pp. 403-506.

and engineering data, 41(4), pp. 876-979.

Transfusion and apheresis science, 34(3), pp. 271-287.

During Addition and Removal of Cryoprotective Agents: Theory and Technology 135

Muldrew, K. and McGann, L.E. (1994) The osmotic rupture hypothesis of intracellular

Myrthe, T.W., and Barry A. B. (2004) Step-wise dilution for removal of glycerol from fresh

Noiles, E.E., Mazur, P., Watson, P.F., Kleinhans, F.W. and Crister, J.K. (1993) Determination

Papanek, P. T., 1978, "The water permeability of the human erythrocyte in the temperature

Penninchx, P., Poelmans, S., Kerremans, R. and De Loecher, W. (1984) Erythrocyte

Polge, C. Smith, A.U. and Parkes, A.S. (1949) Revival of spermatozoa after vitrification and

Preston, G.M., Carroll, T.P., Guggion, W.B., and Agre, P.(1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP 28. Science 256, 385-387. Rowe, A.W., Eyster, E. and Kellner, A. (1968) Liquid nitrogen preservation of red blood

Sherman, J.K. (1973) Synopsis of the use of frozen human sperm since 1964: state of the art of

Spector, P.C., Goodnight, J.H., Sall, J.P., Sarle, S.W. and W.M. Stanish (1985) The GLM and

Steponkus, P.A. and Wiest, S.C. (1979) Freeze-thaw induced lesions in the plasma

Ternstrom, G., Sjostrand, A., Aly, G., and Jernqvist, A. 1996, "Mutual Diffusion Coefficients

Valeri, C. R., 1975, "Simplification of the methods for adding and removing glycerol during

Valeri, C. R., and Ragno, G., 2006, "Cryopreservation of human blood products,"

Valeri, C. R., Ragno, G., Pivacek, L., and O'Neill E. M., 2001, "In vivo survival of apheresis

and cryopreserved equine spermatozoa. Animal Reproduction Science 84, 147-

of water permeability coefficient for human spermatozoa and its activation energy.

swelling after rapid dilution of cryoprotectants and its prevention. Cryobiology.

cells for transfusion: a low glycerol-rapid freeze procedure. Cryobiology, 5,

the Catmod procedure. In SAS Institute, Inc., SAS User's Guide: Statistics, 5th end.

membrane. In Lyons, J.M., Graham, D. and Raison, J.K. (eds), Low Temperature Stress in Crop Plants: The rolle of the Membrane. Academic Press, New York, pp.

of Water + Ethylene Glycol and Water + Glycerol Mixtures," Journal of chemical

freeze-preservation of human red blood cells with the high or low glycerol methods: biochemical modification prior to freezing," Transfusion, 15(3), pp. 195-

RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4 degrees C in AS-3 for up to 21 days," Transfusion, 41(7), pp.


Katkov, I.I. (1998b) Cell suspensions in high concentrations of a permeable cryoprotectant: Optimization of addition and dilution protocols. Cryobiology 37, 403-404 Kedem, O. and Katchalsky, A. (1958) Thermodynamic analysis of the permeability of biological membrane to nonelectrolytes. Biochim. Biophys. Acta, 27, 229-246. Kleinhans, F. W., 1998, "Membrane Permeability Modeling: Kedem-Katchalsky vs a Two-

Kleinhans, F.W., Travis, V.S., Du, Junying, Villines, P.M., Colvin, K.E. and Criter, J.K. (1992)

Leibo, S.P. (1981) Preservation of ova and embryos by freezing. In Brackett, E.G. Seidel, G.E.

Leibo, S.P. (1986) Cryobiology: preservation of mammalian embryos. Basic Life Sci., 37, 251-

Leibo, S.P. and Mazur, P. (1978) Methods for the preservation of mammalian embryos by

Mazur, P. (1984) Freezing of living cells: mechanism and implications. Am. J. Physiol. 247,

Mazur, P. and Leibo, S.P. (1977) Mechanisms of freezing damage in bacteriophage T4

Mazur, P. and Schneider, U. (1984) Osmotic consequences of cryoprotectant permeability

Mazur, P. and Schneider, U. (1986) Osmotic responses of preimplantation mouse and boving embryos and their cryobiological implications. Cell Biophys., 8, 259-284. Mazur, P., Leibo, S.P. and Chu, E.H.Y. (1972) A two-factor hypothesis of freezing injury.

McGann, L.E., Turner, A.R. and Turc, J.M. 91982) Microcomputer interface for rapid

Meryman, H.T. (1970) The exceeding of a minimum tolerable cell volume in hypertonic

Meryman, H. T., 2007, "Cryopreservation of living cells: principles and practice,"

Meryman, H. T., and Hornblower M., 1972, "A method for freezing and washing red blood cells using a high glycerol concentration," Transfusion, 12(3), pp. 145-156. Meryman, H. T., and Hornblower, M., 1977, "A simplified procedure for deglycerolizing

Measurement of human sperm intracellular water volume by electron spin

and Seidel, S.M. (eds), New Technologies in Animal Breeding Academic Press,

freezing. In Daniel, J.C., Jr (ed.), Methods in Mammalian Reproduction. Academic

(Discussion). In Elliott, K. and Whelan, J. (eds), The Freezing of Mammalian Embryos. Ciba Foundation Symposium No. 52. Elsevier, Amsterdam, pp.

and its relation to the survival of frozen-thawed embryos. Theriogenology, 21, 68-

measurement of average volume using an electronic particle counter. Med. Biol.

suspension as a cause of freezing injury. In Wolstenholme, G.E.W. and O'Connor, M. (eds), The Frozen Cell. CIBA Foundation Symposium. Churchill, London, pp.

red blood cells frozen in a high glycerol concentration," Transfusion, 17(5), pp.

Parameter Formalism," Cryobiology, 37(4), pp. 271-289.

resonance. J. Androl. 13, 498-506.

New York, pp. 127-139.

Press, New York, pp. 179-201.

Exp. Cell Res. 71, 345-355.

Eng. Comput., 20, 117-120.

Transfusion, 47, pp. 935-945.

272.

C125-C142.

255-226.

79.

51-67.

438-442.


**Part 2** 

**Stem Cells and Cryopreservation** 

**in Regenerative Medicine** 

