Strikha O. Effect of cryoprotectants and cryopreservation on electrical insulating properties of mouse oocytes and 2-cell embryos cytoplasmic membranes

The research object was the variation of cell membrane potential due to effect of ethylene glycole and dimethyl sulfoxide solutions and cryopreservation. The research aim was to ascertain the conditions of electrical insulating properties distortion (electrical breakdown) observed in mouse oocytes and 2-cell embryo cytoplasmic membranes during low-temperature preservation. Methods: physico-mathematical and computer simulation, pulse conductometry, hormonal superovulation, optical microscopy and statistical processing of the results. The equipment comprised an instrumental complex for electroporation, and a light microscope MBS-9. The original physical and mathematical model of transmembrane mass transfer between cells and surrounding cell solution during freezing cell suspension has been developed. The model involves optimal cooling rates, geometrical and transport characteristics of cells, composition of cryoprotective medium and allows estimating the changes in cell membrane potential. For the first time it has been shown that slow cooling of mouse oocytes and 2-cell embryo (<5 К/мин) led to membrane hyperpolarization alternated by depolarization, while rapid freezing resulted in reaching of membrane potential the range -50 to -80 mV, that might cause electrical breakdown. It has been established that 1,5 min long exposure of two-cell embryos in the ethylene glycol and sucrose medium did not lead to an irreversible electrical breakdown and blastomere lysis either prior to or after cooling of cells down to 77 K. Embryo resistance to electrical breakdown was violated following 3 min long exposure in ethylene glycol and sucrose medium. It has been shown that reversible electroporation of 2-cell embryo was observed in the range of field intensity of 50 to 300 kV/m. Field intensity of more than 350 kV/m caused the irreversible electrical breakdown. The results could be used for development of new technologies and improvement of existing ones for mammalian embryos cryopreservation and assessment of functional value of cells to be used in various cell biotechnologies. The proposed physico-mathematical model allows to narrow significantly the optimal condition of cryopreservation region of search.