Article

Comparison of dilute and nondilute osmotic equilibrium models for erythrocytes

Authors:
  • Erenkoy physical and Rehabilitation Training and Research Hospital
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Abstract

Successful cryopreservation requires the addition of cryoprotective agents (CPAs). The addition of permeating CPAs, such as glycerol, is associated with some risk to the cells and tissues. These risks are both related to the CPA themselves (CPA toxicity) and to the volume response of the cell (osmotic damage). To minimize the potential for damage during cryopreservation, mathematical models are often employed to understand the interactions between protocols and cell volume responses. In the literature, this volume response is usually captured using ideal and dilute approximations of chemical potential and osmolality, an approach that has been called into question for cells in high concentrations of CPAs. To address this, the relevance of non-ideal and non-dilute models has been explored in a number of cell types in the presence of permeating CPAs. However, it has not been explored in erythrocytes, which have a cytosolic hemoglobin content of more than 20% by volume and are cryopreserved in 40% glycerol. Because hemoglobin has been suggested to be a highly non-ideal solute, if the non-ideal and non-dilute transport model is relevant to any cells, it should be relevant to erythrocytes. Here we investigate the use, and accuracy, of both the dilute and non-dilute models in predicting cell volume changes during CPA equilibration in erythrocytes, and demonstrate that using published values for the non-ideal and non-dilute model, applied to erythrocytes, leads to model predictions inconsistent with experimental data, whereas dilute approximations align well with experimental data.

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The osmotic virial equation was used to predict osmolalities of solutions of interest in biology. The second osmotic virial coefficients, Bi, account for the interactions between identical solute molecules. For multisolute solutions, the second osmotic virial cross coefficient, Bij, describes the interaction between two different solutes. We propose to use as a mixing rule for the cross coefficient the arithmetic average of the second osmotic virial coefficients of the pure species, so that only binary solution measurements are required for multisolute solution predictions. Single-solute data were fit to obtain the osmotic virial coefficients of the pure species. Using those coefficients with the proposed mixing rule, predictions were made of ternary solution osmolality, without any fitting parameters. This method is shown to make reasonably accurate predictions for three very different ternary aqueous solutions: (i) glycerol + dimethyl sulfoxide + water, (ii) hemoglobin + an ideal, dilute solute + water, and (iii) bovine serum albumin + ovalbumin + water.
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To cryopreserve human ovarian tissue using solid-surface vitrification (SSV) technique for the first time. Human ovarian slices from each of 26 patients were randomly allocated to fresh, SSV, and slow-freezing groups, respectively. Histological observation and the TUNEL assay of the tissue were performed after cryopreservation. In vitro culture was done to study the initial recruitment of follicles and hormone production ability after SSV/slow-freezing. The majority of primordial follicles were maintained intact through either SSV or the slow-freezing method. No statistically significant destructive effect of SSV or slow-freezing for primordial follicles and stromal cells was found using the TUNEL assay. In the SSV and slow-freezing groups, estradiol and progesterone were secreted continuously during 10 days in culture, and the proportions of growing follicles increased significantly comparing to the uncultured fresh group. The follicular proportions and the concentrations of estradiol and progesterone exhibited no statistically significant differences between the SSV and slow-freezing groups. SSV is an effective, simple and inexpensive alternative for human ovarian tissue cryopreservation.