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TECHNICAL PAPERS: Fluids/Heat/Transport

Membrane Transport Properties of Equine and Macaque Ovarian Tissues Frozen in Mixtures of Dimethylsulfoxide and Ethylene Glycol

[+] Author and Article Information
A. Kardak

Bioengineering Laboratory, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803

S. P. Leibo

Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148; Audubon Center for Research of Endangered Species (ACRES), New Orleans, Louisiana 70131

R. Devireddy1

Bioengineering Laboratory, Department of Mechanical Engineering,  Louisiana State University, Baton Rouge, LA 70803devireddy@me.lsu.edu

1

Corresponding author.

J Biomech Eng 129(5), 688-694 (Feb 14, 2007) (7 pages) doi:10.1115/1.2768107 History: Received May 22, 2006; Revised February 14, 2007

Abstract

The rate at which equine and macaque ovarian tissue sections are first cooled from $+25°Cto+4°C$ has a significant effect on the measured water transport when the tissues are subsequently frozen in $0.85M$ solutions of glycerol, dimethylsulfoxide (DMSO), or ethylene glycol (EG). To determine whether the response of ovarian tissues is altered if they are suspended in mixtures of cryoprotective agents (CPAs), rather than in solutions of a single CPA, we have now measured the subzero water transport from ovarian tissues that were suspended in mixtures of DMSO and EG. Sections of freshly collected equine and macaque ovaries were suspended either in a mixture of $0.9M$ EG plus $0.7M$ DMSO (equivalent to a mixture of $∼5%$$v∕v$ of EG and DMSO) or in a $1.6M$ solution of only DMSO or only EG. The tissue sections were cooled from $+25°Cto+4°C$ and then frozen to subzero temperatures at $5°C∕min$. As the tissues were being frozen, a shape-independent differential scanning calorimeter technique was used to measure water loss from the tissues and, consequently, the best fit membrane permeability parameters ($Lpg$ and $ELp$) of ovarian tissues during freezing. In the mixture of $DMSO+EG$, the respective values of $Lpg$ and $ELp$ for equine tissue first cooled at $40°C∕min$ between $+25°C$ and $+4°C$ before being frozen were $0.15μm∕minatm$ and $7.6kcal∕mole$. The corresponding $Lpg$ and $ELp$ values for equine tissue suspended in $1.6M$ DMSO were $0.12μm∕minatm$ and $27.2kcal∕mole$; in $1.6M$ EG, the values were $0.06μm∕minatm$ and $21.9kcal∕mole$, respectively. For macaque ovarian tissues suspended in the mixture of $DMSO+EG$, the respective values of $Lpg$ and $ELp$ were $0.26μm∕minatm$ and $26.2kcal∕mole$. Similarly, the corresponding $LLg$ and $ELp$ values for macaque tissue suspended in $1.6M$ DMSO were $0.22μm∕minatm$ and $31.4kcal∕mole$; in $1.6M$ EG, the values were $0.20μm∕minatm$ and $27.9kcal∕mole$. The parameters for both equine and macaque tissue samples suspended in the $DMSO+EG$ mixture and first cooled at $0.5°C∕min$ between $+25°C$ and $+4°C$ were very similar to the corresponding values for samples cooled at $40°C∕min$. In contrast, the membrane parameters of equine and macaque samples first cooled at $0.5°C∕min$ in single-component solutions were significantly different from the corresponding values for samples cooled at $40°C∕min$. These results show that the membrane properties of ovarian cells from two species are different, and that the membrane properties are significantly affected both by the solution in which the tissue is suspended and by the rate at which the tissue is cooled from $+25°Cto+4°C$ before being frozen. These observations suggest that these variables ought to be considered in the derivation of methods to cryopreserve ovarian tissues.

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Figures

Figure 3

Volumetric response of macaque ovarian tissue as a function of subzero temperatures using the DSC technique at a freezing rate of 5°C∕min. (a) and (b) show the water transport response obtained in the presence of 1.6M solutions of DMSO and EG, respectively. In both figures, the water transport data from the ovarian tissue cooled at 0.5°C∕min between +25°C and +4°C or at 40°C∕min between +25°C and +4°C are shown as filled (•) and open (엯) circles, respectively. The Krogh model simulated response using the predicted best fit membrane permeability parameters (Table 2) in Eqs. 1,2 are also shown (—). The nondimensional volume is plotted along the y axis and the subzero temperatures are shown along the x axis. The error bars represent the standard deviations in the data (n=6).

Figure 2

Volumetric response of equine ovarian tissue as a function of subzero temperatures using the DSC technique at a freezing rate of 5°C∕min. (a) and (b) show the water transport response obtained in the presence of 1.6M solutions of DMSO and EG, respectively. In both figures, the water transport data from the ovarian tissue cooled at 0.5°C∕min between +25°C and +4°C or at 40°C∕min between +25°C and +4°C are shown as filled (•) and open (엯) circles, respectively. The Krogh model simulated response using the predicted best fit membrane permeability parameters (Table 2) in Eqs. 1,2 are also shown (—). The nondimensional volume is plotted along the y axis and the subzero temperatures are shown along the x axis. The error bars represent the standard deviations in the data (n=6).

Figure 1

Volumetric response of equine (a) and macaque (b) ovarian tissue as a function of subzero temperatures using the DSC technique at a freezing rate of 5°C∕min. The samples were frozen in the presence of 0.9M EG and 0.7M DMSO. In both figures, the water transport data from the ovarian tissue cooled at 0.5°C∕min between +25°C and +4°C or at 40°C∕min between +25°C and +4°C are shown as filled (•) and open (엯) circles, respectively. The Krogh model simulated response using the predicted best fit membrane permeability parameters (Table 1) in Eqs. 1,2 are also shown (—). The nondimensional volume is plotted along the y axis and the subzero temperatures are shown along the x axis. The error bars represent the standard deviations in the data (n=9).

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