Kyryliuk G. Influence of cryoprotectants and freezing regimens on mechanical damage of cells within subeutectic interval of temperatures

The thesis is devoted to studying the cryoprotectants' action (DMSO, glycerol, PEO-1500) and freezing regimens on mechanical damage of biological objects (Saccharomyces cerevisiae, cells of diploid human cultures and human fibroblast cells) within sub-eutectic interval of temperatures during their cryopreservation. The technique of determining of mechanical characteristics of samples by consecutive loadings has been developed on the basis of a method of thermoplastic deformation. By means of this technique the researches of elastic-plastic properties of cryoprotective solutions near to the temperatures of their glasstransition are carried out. The new approach to definition of optimum concentration of cryoprotective substances is offered at their application during cryopreservation. There are established the threshold concentrations of the studied cryoprotective solutions, at which closed liquid-phase inclusions transform through layers. During cryopreservation of S. cerevisiae cells and those of humancultures there was observed an abrupt increase in cell viability at reaching the threshold concentrations of cryoprotectants in their solutions after freezing and thawing, pointing to general character of the revealed phenomenon. By means of the method of thermoplastic deformation the sub-eutectic temperature intervals and the vitrification ones for the solutions of cryoprotectants have been found. It has been established that these temperature intervals should be taken into account during cryopreservation for lessening mechanical damage of biological objects due to plastic deformation of pressures in closed inclusions at the non-equilibrium crystallization. Taking into account the obtained experimental data there was optimized the freezing regimens of biological objects on the example of S. cerevisiae cells.