Kholodnyy V. Investigation of processes, occurring on various stages of low temperature preservation in the suspensions of nucleated cells of bone marrow and cord blood

The thesis is devoted to the studying of the processes of mass exchange of the cells of bone marrow and cord blood with an environment at the main stages of cryopreserevation and the determining of the relationship of these processes with the damage of cells due to the effect of basic cryodamage factors: intracellular crystallization, dehydration and rehydration. Using the methods of light microscopy, physical and mathematical modeling, and cryomicroscopy there were established the peculiarities in osmotic behaviour of the nucleated cells of murine bone marrow and human cord blood at all main stages of cryopreservation (exposure of cells in cryoprotective medium, cooling, freeze-thawing, and removal of cryoprotectant out of cells). There were found the stipulating cryoprotection and cryodamage parameters, determining the processes of mass transfer for water and commonly used cryoprotectants (DMSO, 1,2- propane diol, glycerol) in the "cell-environment" system. Using the methods of supravital staining, analysis of qualitative and quantitative composition of cellular suspension, adhesive ability and phagocyte activity there were determined the admissible limits of the change in cell volume which do not result in a significant reduction in viability. It is established that plasma membrane permeability to water does not depend on the appearance of the above stated cryoprotectants in the solution. The studied cryoprotectants formed the following row for increase in its permeability through membranes of nucleated cells of bone marrow and cord blood: glycerol>dimethyl sulfoxide>1,2-propane diol. These permeability coefficients do not depend on the orientation of the transmembrane flow of water and cryoprotectants. Bone marrow and cord blood cells are resistant to osmotic effects, leading to the changes in its volume within the range of 60-120% in respect of the value under isotonic conditions. They survive well the volume changes, resulted from one-step dilution with dimethyl sulfoxide and 1,2-propane diol down tothe final concentration of 1 M, and glycerol down to that of 0,75 M. Characteristic indices of cells' damaging after dehydration are the cytoplasm vacuolisation, appearance of transmembrane defects and cell swelling. In the case of unprotected cells the intracellular crystallisation leads to membranes ruptures and the loss of cell content. The presence of cryoprotectants diminishes the manifestation rate of these damages. The preventing of intracellular crystal formation by means of decrease in the level of extracellular overcooling in the cryoprotected suspensions is essential for a high rate of cell integrity. Cell volume increase over the initial values at the stage of one-step washing of cryoprotectant does not always lead to a immediate damage of membrane barrier functions for extracellular solutes. The advantage of the two-step cryoprotectant washing out of cell suspension is the extra-increase of the volume, which in this case does not exceed 125% of initial value (in contrast to 170-200% at one-step removal of cryoprotectant). Hypertonic concentration use of non-penetrating to a cell substance in the washing-out medium allows a complete prevention of cell volume rise.