Mìkrobìologìâ ì Bìotehnologìâ (Dec 2015)
STUDY OF CRYOINJURIES OF FREE AND IMMOBILIZED IN SODIUM ALGINATE GEL SACCHAROMYCES CEREVISIAE YEAST CELLS
Abstract
The aim of this study was the revealing of the peculiarities of cryoinjuries of free and immobilized in sodium alginate gel Saccharomyces cerevisiae cells after cryopreservation. Methods. The research object was the Saccharomyces cerevisiae yeast cells. By means of fluorescent dyes Annexin V and 7AAD with flow cytometry the injuries of frozen-thawed Saccharomyces cerevisiae cells were assessed. Results. The characteristic features of negative effect of low temperatures were impairments in the integrity and lipid asymmetry of cytoplasm membranes (CPM), DNA fragmentation. The performed analysis of asymmetric distribution of phospholipids in membrane of Saccharomyces cerevisiae yeast cells has shown that the rearrangements in lipid membrane structure during cryopreservation may occur not only under the effect of physical and chemical factors but also due to cryoprotectants. The yield of phosphotidylserine in an outer monolayer layer of CPM was observed in 2.5% of immobilized cells (group 2). The number of Annexin V – labeled cells in group 4 was twice higher relative to the amount of intact cells after freezing. A similar tendency was observed with regard to this kind of damage in the samples of free cells. However, it should be noted that the number of AnnexinV – labeled cells in group 1 was significantly lower and it is comparable with the controls. We believe that the reduction in the number of cells with disordered asymmetric distribution of phospholipids in group 1 was due to increase in the number of completely destroyed (not subjected to analysis) cells in these samples. It has been found that all the tested samples, including controls, there were the cells being at different stages of apoptosis/necrosis. The difference in the samples consists in the value of the percentage of cells eliminated from the analysis of flow cytometry. The number of completely destroyed cells in immobilized samples was significantly lower than in the group of free cells in a suspension. It has been established that there are the cells being at different stages of apoptosis, in all the studied samples including the control ones. The difference in the samples consists in the value of the percentage of the cells eliminated from the process of flow cytometry analysis. The number of completely destroyed cells in immobilized samples was statistically and significantly lower than in the groups of free in suspension cells. Conclusions. It has been established that under the influence of cryopreservation damaging factors in a part of free and immobilized in alginate gel S. cerevisiae cells there were developed the disorders in an integrity and lipid asymmetry of the cytoplasmic membranes, DNA fragmentation. The presence of DMSO as a cryoprotectant in preserving medium has been shown to lead to an increase in the number of cryopreserved cells with an asymmetric distribution of phospholipids in cell membranes. The number of cells in early apoptosis after freezing with DMSO as a cryoprotectant in 2–2.5 times exceeded the number of similar cells frozen without cryoprotectant. Cryopreservation of yeasts in polysaccharide gel of sodium alginate may be an alternative way to preservation of biological objects under the protection with traditional cryoprotective agents, allowing a high degree of viability and preservation of cells with no cryoprotectants.
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