Cryopreservation of Equine and Bovine Erythrocytes Using Combined Protective Media

The cryopreservation eff ectiveness for equine and bovine erythrocytes with the use of one component (20% DMSO) and combined (10% DMSO, 20% PEO-1500) protective medium was comparatively analyzed. Using fl uorescence microscopy and fl ow cytometry, the 3-DAB fl uorescent dye has been shown to stain the membranes of red blood cells and can be used to evaluate their condition at all cryopreservation stages. It was found that the use of combined protective medium can signifi cantly improve the results of cryopreservation of equine and bovine red blood cells in comparison with one-component media. By cytometric data it has been established that the parameters of equine erythrocytes after freezing-thawing in DMSO-based medium did not correspond the control even after cryoprotectant removal, but after freezing-thawing in combined cryoprotective medium and cryoprotectant washing-out they corresponded to the control. The increased fl uorescence in equine and bovine erythrocytes membranes and changes in cytograms after mixing the cells with 20% DMSO suggested its strong membranotropic infl uence. Hemolysis level after applying a combined cryoprotective medium of equine and bovine erythrocytes was signifi cantly lower at all cryopreservation stages than that when 20% DMSO was applied.

Animal blood components are often used in veterinary medical practice during treatment of intoxications, signifi cant blood loss, immune system disorders, etc. [13,17]. Looking for a donor animal with a corresponding blood group and testing this blood for the absence of blood-parasitic and infectious diseases [10,11] require a long time, therefore, in urgent cases, it is important to have the blood stocks with the possibility of long-term storage in cryobanks [9]. It has been shown that in contrast to cryopreservation of human red blood cells, the freezing of the cells of most animal species using protective media based on glycerol or 1,2-propanediol is ineff ective [6,12,15]. The cryoprotectant dimethylsulfoxide (DMSO) showed a higher protective eff ect on erythrocytes of horses, bulls, cats and dogs, but the level of hemolysis of the cells after all the stages of cryopreservation was quite a high [6,12]. Therefore it is necessary to fi nd more eff ective media for cryopreservation of animal erythrocytes. The use of exocellular cryoprotective compounds results in a low level of erythrocyte hemolysis after thawing, but high osmotic fragility rates do not allow them to be used for hemotransfusion [6]. It has been found that in order to freeze the cells of certain species of animals is more suitable is the combination of protective media based on endo-and exocellular compounds [12,14].
Currently, in cryobiology there are widely used the fl uorescence methods of cell analysis, fl ow cytometry and luminescent microscopy using fl uorescent dyes that respond to changing microenvironment and have high solvatochromic indices as well as a signifi cant increase in quantum yield during binding to living macromolecules [4,7,16]. So, a fl uorescent dye 3-DAB (3-dimethylaminobenzanthrone) is a neutral substance which is practically insoluble in water and is sensitive to polarity of the environment (solvatochromic eff ect), possesses suffi cient hydrophobicity to penetrate into biological objects and non-covalently bind with their biomacromolecules [7]. It is important that 3-DAB has a high photochemical stability and slight quantum yield of fl uorescence in aqueous media, sharply increasing in hydrophobic phase, in concentrations of 10 -4 -10 -3 M it does not toxically aff ect the cells [4].
The research aim was to compare the eff ectiveness of single-component and combined cryoprotective media to cryopreserve the equine and bovine red blood cells.
To freeze the samples, two cryopreservation media, prepared with phosphate-buff ered saline (0.15 M NaCl, 5 mM sodium phosphate buff er, pH 7.4) were used: 1 -20% DMSO; 2 -10% DMSO, 20% polyethylene oxide-1500 (PEO-1500). The cryopreservation medium was added to the washed erythrocytes by droplets in a 1: 1 ratio and incubated for 15 minutes at room temperature. Samples were frozen in 4.0 ml polystyrene tubes by immersion into liquid nitrogen, heated in a water bath (42°C) until a liquid phase appeared. From the cryopreservation medium, the cells were once washed with 0.6 M NaCl and twice with 0.15 M NaCl on 5 mM sodium phosphate buff er, pH 7.4. The level of hemolysis at all stages of cryopreservation was measured with a spectrophotometer 'Pye Unicam SP 8000' ('Pye Unicam Ltd', UK) and expressed as a percentage of 100% of hemolysed cells.
For the staining of red blood cells, 3-DAB (SETA BioMedicals, USA) fl uorescence dye was used at a concentration of 40 μM for microscopic studies and at 4 μM for cytometric ones [7].
Cells were incubated with dye for 15 min and washed from excess dye by centrifugation at 1300g for 3 min. Fluorescent images of erythrocytes were obtained with fl uorescent microscope Axio-ObserverZ1 (Carl Zeiss, Germany). The fl uorescence characteristics of red blood cells, stained with probes, were studied using a fl ow cytometer 'FACS Calibur' (Becton Dickinson, USA). Fluorescence was excited by light with a wavelength of 488 nm (argon laser). Each experiment was repeated fi ve times.
Після інкубації еритроцитів у розчині ДМСО посилюються флуоресценція частини клітин і зменшується їх діаметр (рис. 1, 2). Це може бути пов'язане зі зміною форми клітин, близькою до at diff erent stages of cryopreservation. It is apparent that 3-DAB stains the membranes of mammalian erythrocytes, so it can be used to evaluate their state. Localization of the probe in membranes and intracellular structures varies depending on the microenvironment of its molecules. The 3-DAB molecules, due to hydrophobic properties, are assumed to be concentrated at the border of nonpolar area of cell membrane lipids [4,7]. The binding of the dye to membrane and intracellular structures probably is due to a non-covalent hydrophobic way, and the place of its localization is nonpolar areas ('pockets') near the macromolecules. The 3-DAB dye partially penetrates into intracellular medium of red blood cells, yeast, human sperm, dogs, cattle, etc., but because of high water content, fl uorescence from the probe molecules in this area is negligible.
After an incubation of erythrocytes in DMSO solution, the fl uorescence of some cells is increases and the diameter of cells decreases (Fig. 1, 2). This may be due to a change in the shape of cells close to the spherical, and an increase in the permeability of membranes of some cells to the dye. The number of hydrophobic binding sites increases and, since the 3-DAB dye interacts with biological structures by hydrophobic mechanism, the fl uorescence of erythrocytes is signifi cantly enhanced. Such an eff ect was observed by I.A. Buriak et al. [4] during a partial damage to membranes, with a complete cell injury resulting in fl uorescence quenching by water.
Додавання до суспензії еритроцитів коня і бика кріозахисного середовища, що містить сполуки Freezing-thawing of equine and bovine erythrocytes in a single-component medium causes the appearance of erythrocytes with a homogeneous bright color (Fig. 1, 2). Probably, increasing the permeability of membrane during its damage leads to a more vivid staining of cells. After all the stages of cryopreservation (freezing-warming-washing) of equine erythrocytes in DMSO solution no cells with a bright fl uorescence were observed. They may be removed from the suspension after cryoprotectant washing. Percentage of dead cells at all the stages of cryopreservation was determined using the level of hemolysis. Fig. 3 shows that at the stage of washing from the cryoprotectant, the level of hemolysis of erythrocytes increases by almost 30% if compared to that in the samples immediately after freezingwarming. This suggests that after washing-out the cells, which were partially damaged at the stage of incubation with cryoprotectant and freezingwarming, were completely destroyed.
Adding to the suspension of equine and bovine erythrocytes of cryoprotective medium containing compounds of diff erent type of action (DMSO and PEO-1500) did not substantially enhance the fl uorescence of erythrocyte membranes, in contrast to a single-component solution based on DMSO. This indicates a less membranotropic eff ect of the combined medium if compared to a single-component one based on DMSO. After all the stages of cryopreservation in the combined medium, the erythrocyte sizes and intensity of the fl uorescence of membranes did not diff er signifi cantly from the control (see Fig. 1, 2).
The distribution of cells in cytogram is known to be infl uenced by morphological changes of red blood cells [18]. So N.G. Zemlyanskykh et al. [19], according to microscopic observations and cytogram analysis, revealed the correlations between the shift of cell division in the diagram to the right and the increase of sphericity of red blood cells. These changes were reversible until the point of hemolysis was reached, and after returning to isotonic conditions, the shape of most erythrocytes and their cytogram were back to the control parameters. Based on the obtained cytometric data, we found that for the equine erythrocytes, cryopreserved with DMSO, the cytogram was not restored, even after washing-out the cryoprotectant. This fact indicates irreversible changes in the shape of erythrocytes, therefore their normal functional activity after transfusion is impossible.
Зниження токсичної дії ДМСО можливе при зменшенні його концентрації, часу та температури інкубації. Комбінація ДМСО з кріопротекторами іншого типу дії дозволяє знизити його концентрацію у розчині і відповідно зменшити ток-DMSO than in those of a bull and dog. After cryopreservation with PEO-1500 the hemolysis in equine erythrocytes was, in contrast, smaller, that evidenced to their sensitivity to specifi c toxic eff ects of DMSO. We recorded a signifi cantly higher hemolysis level in the presence of DMSO for equine erythrocytes relative to the ones of a bull (see Fig. 3). In the combined medium this diff erence was not found.
B.P. Best showed [3] that DMSO at a concentration of 10% had a toxic eff ect on cells, e. g. led to changes in the membranes of hamster fi broblasts, caused irreversible ultrastructural damage to the myocardium of rats, reduced the clonogenic potential of peripheral blood precursors etc. Moreover, in addition to osmotic eff ects, there is a direct blocking eff ect of DMSO molecules on the proteins of the membrane channels, and its hydrophilicity and ability to destabilize the conformation of the protein increase with a rise in temperature. In addition, DMSO is also characterized with a specifi c damaging eff ect, i. e. cell membrane toxicity [3]. However, despite this, DMSO is widely used in cryopreservation of many biological objects due to high permeability and vitrifying ability. Modifi cation of water structure with DMSO molecules, which leads to the tendency of liquid overcooling and formation of the vitrifi cation phase, is due to the fact that the time of existence of hydrogen bond DMSO-water is several times longer than that of the hydrogen bond water-water. The hydrogen bonds of the sulfi nyl group of DMSO with water are stronger (about 30 kJ/mol) than the hydrogen bonds between water molecules (about 20 kJ/mol). With temperature increase the interaction of DMSO with water becomes weaker [1].
Reducing the toxic eff ect of DMSO is possible by decreasing its concentration, time and incubation temperature. A combination of DMSO with other type of cryoprotectants can reduce its concentration in the solution and, accordingly, diminish the toxic eff ect. In this case, the total concentration of cryoprotective compounds is maintained, suffi cient for modifying the water structure during freezing [8].
3-DAB stains membranes of equine and bovine erythrocytes, that allows to observe the changes in the state of cells at diff erent stages of cryopreservation. It has been established that due to the damaging factors of the cryopreservation of the membrane, the part of the erythrocytes acquires a loose structure, and the number of hydrophobic sites of the probe binding increases, which causes the strengthening of fl uorescence of the cells with damaged membranes. This fact is due to the pos-