ИСТИНА

Cryoconservation is one of the key procedure for the preserving of genetic material. Two general approaches are suggested for this process- slow freezing and vitrification. Despite some progress in research of vitrification of cells the main technique for the preserving of spermatozoids is slow freezing. This procedure involves consequent steps of cooling of the sample to 4 °C, incubation with cryoprotectant, cooling down to storage temperature. Two major side effects leading to death of cell should be avoided during cryoconservation- formation of ice crystals and osmotic shock of cell membrane. Due to high sensitivity of spermatozoids membranes to osmotic stress, the choice of cryoprotectant is still challenging. Oligosaccharides like cyclodextrins (CDs) were found to be effective protectants for the spermatozoids but on the other hand CDs were shown to possess relatively high cyctotoxicity due to extraction of cholesterol (Ch) molecules from cell membranes. In or work we suggest to use the complexes of cyclodextrins with cholesterol as cryoprotectors. It was found that CD/Ch complexes have reduced cytotoxicity, but retain possibility to suppress ice crystals formation. Moreover, CD/Ch complexes were demonstrating to interact with cell membrane enriching it with Ch molecules. As a result, spermatozoid membranes became more tolerate to osmotic stress during freezing process. In order to increase the fraction of Ch in cell membranes the cryoprotectant with high volume fraction of Ch was developed. Electrostatic adsorption of anionic carboxymethyl cyclodextrins (CMCDs) -on the surface of cationic liposomes resulted in formation of colloid stable particles representing individual liposome decorated by numerous CDs molecules. By means of laser microelectrophoresis, light scattering, Langmuir monolayers study atomic force microscopy it was stated that CMCDs are adsorbed on liposomal membrane forming associates of tens of CDs. Incorporation of hydrophobic molecules into inner cavity of CMCDs does not affect on adsorption and structure of interfacial complexes. By means of fluorescent microscopy it was shown that CMCDs/liposomes complexes are capable to be effectively adsorbed on model cell membranes. Taking into account low cytotoxicity of CMCDs and their complexes with liposomes the ensembles of anionic CDs on cationic liposomes look promising for utilization as cryoprotectors for spermatozoids in slow freeze cryoconservation process. The research is being conducted within the framework of the Interdisciplinary Scientific and Educational School of Moscow State University "Cosmos", the project "Improving the efficiency of cryopreservation of biological material for long-term storage and transportation over long distances, including in space conditions". Young scientist Ilya Grigoryan thanks for the support the Foundation for the Development of Theoretical Physics and Mathematics "BASIS”.