Direct Measurement of Cell Volume Osmotic-Induced Changes in Spermatozoa Used for Artificial Cryopreservation

Factors like thawing-induced rehydration and freezing-induced dehydration cause cellular damage and induce anisotropic mechanical strains on macromolecules and membranes. The determination of cell volume changes can be associated with cellular thawing and freezing damage. Therefore, analysis of cell volume osmotic-induced changes can help improve cryopreservation processes of cells.

Freezing and Thawing of Cell Suspensions

Cooling rates can be fast or slow depending on how rapidly the heat leaves the cells. The extracellular solution often freezes first. In the remaining unfrozen extracellular water, the extracellular solutes are concentrated, and when cooling rates are sufficiently slow, the cell is dehydrated by osmosis as water enters into the concentrated external solution from the cytoplasm.

In artificial cryopreservation, high and very high cooling rates are used. As a result, sufficient time is not available for most of the water to exit the cell and thus leads to supercooling with minimal osmotic contraction. Supercooling can result in the formation of intracellular ice, which can be quite dangerous. When cooling is sufficiently fast and when the intracellular solution is viscous and concentrated, this will lead to vitrification of the cytoplasm.

However, vitrification seldom occurs because the total internal solute concentration of organisms and cells is not necessarily always high enough. Nevertheless, vitrification can be increased in two ways: either by osmotic contraction or by introducing solutes to the suspending medium that are capable of entering into cell membranes. For example, in an aqueous glass, water molecules do not exist in stable state.

This is because the high viscosity inhibits these molecules from moving around and prevents them from finding their stable, crystalline state. However, when the glass is warmed, freezing can occur. As the temperature rises, but within the equilibrium freezing temperature, the glass’ viscosity decreases and its molecular motion increases. This enables the molecules to rotate and move and occasionally to form ice crystals.

Osmotic-Induced Volume Changes in Spermatozoa

Given the fact that phase transitions are influenced by low temperature, temperature can possibly moderate the osmotic tolerance of sperm and could lead to a lower tolerance range well below phase transition temperatures. In order to acquire an indirect estimation of cell volume, cell permeability can be determined.

Differential Scanning Calorimetry (DSC) can be used to determine the permeability in macaque sperm at sub-zero temperatures. This method has been employed to differentiate the optimal cooling rate for spermatozoa. The DSC technique determines the heat releases during a phase change as a virtue of temperature and time at controlled cooling rates. The DSC method is based on determining the change in integrated heat release between the preliminary freezing of cells, and the final heat release from freezing of lysed cells.

This value is relative to the amount of osmotically active cell water in the sample before freezing. Moreover, the temperature dependence of the heat release variation regulated by the total heat is proportional to the normalized volume of cell water leaving the cells. This makes it possible to measure the volume of sperm cells using the heat releases determined by the DSC technique. A mathematical formula can then be used to calculate the relationship of the cell volume with regard to temperature.

With the help of the Multisizer™ 3, the cell volume can be measured quickly and easily. Changes in cell volume are determined at the time of an osmotic challenge of a cell suspension. The pulse data produced during the study reveals the cell volume within the analysis.

Methods and Materials

First, the sperm cells were measured using the Mulitisizer™ 3 Coulter Counter from Beckman Coulter®. A 100µm aperture tube was then employed and the analyses were performed for a period of 20s. A round-bottom beaker was used to prepare a cell suspension and analysis was performed to ascertain the stability of cell volume. A known quantity of water was then introduced to the cell suspension and the analysis was again carried out to ascertain how a change in osmolality of the media influences the cell volume.

Results and Discussion

Figure 1 illustrates the pulse graph for the control cell volume. It can be seen that throughout the analysis, the cell volume remains stable. Figure 2 shows the effect in cell volume owing to change in media osmolality.

Pulse graph for control cell volume

Figure 1. Pulse graph for control cell volume

Effect in cell volume due to change in osmolality of the media

Figure 2. Effect in cell volume due to change in osmolality of the media

The cell volume at the time of analysis can be known by clicking on the graph, as shown in Figure 3. The Multisizer™ 3 software shows a complete list of cell volume versus time.

Cell volume at any time within in the analysis

Figure 3. Cell volume at any time within in the analysis

Besides the mean cell volume of sperm at different periods of time, the size distribution of the normal cell population at different intervals of time was also obtained. The size distribution for 3 to 4 seconds and 15 to 20 seconds intervals are shown in Figures 4 and 5, respectively. Table 1 summarizes the mean cell volume for every 2 seconds during the 20-second analysis.

The size distribution for 3 to 4 seconds and 15 to 20 seconds intervals, respectively.

The size distribution for 3 to 4 seconds and 15 to 20 seconds intervals, respectively.

Figures 4 and 5. The size distribution for 3 to 4 seconds and 15 to 20 seconds intervals, respectively.

Size Distribution

MCV (Mm3)

Elapsed Time (sec.)

SP 1

22.24

2

SP 2

25.09

4

SP 3

26.77

6

SP 4

27.54

8

SP 5

28.27

10

SP 6

29.08

12

SP 7

29.68

14

SP 8

30.28

16

SP 9

30.66

18

SP 10

31.06

20

Table 1. Mean cell volume (MCV) and different analysis times

Conclusion

The Mulitisizer™ 3 Coulter Counter from Beckman Coulter allows direct and real time measurement of cell volume changes in a fast, simple, and precise way. Analysis of these osmotic-induced changes can help enhance the cell cryopreservation processes.

About Beckman Coulter

Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.


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Last updated: Mar 1, 2019 at 7:59 AM

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