E R Valeeva’s research while affiliated with Russian Academy of Sciences and other places

The idea of using a higher pressure to reduce damage due to freezing has been thoroughly theoretically examined, while there is little experimental evidence on cryopreservation of living systems at a higher hydrostatic pressure. A study was made to assess the viability of HeLa cells at a pressure of 1.0–2.0 kbar, the toxic effects of five classical cryoprotectants under these conditions, and the viability of HeLa cells during slow (conventional) cryopreservation at a pressure of 1.0 or 1.5 kbar. High resistance to higher hydrostatic pressure was experimentally demonstrated for HeLa cells; i.e., 100% cell survival was observed after cell exposure to a pressure of 1.0 kbar for 60 min. The toxic effect of the cryoprotectants increased under pressure in the following order: glycerol < ethylene glycol < 1,2-propanediol = DMSO < DMSO + formamide. Glycerol and ethylene glycol were the least toxic and additionally exerted substantial baroprotective effects. A high cell survival rate of 83 ± 16% was achieved with 10% glycerol used for cryopreservation at a pressure of 1.0 kbar according to fluorescence staining data, but did not exceed the survival rates observed in control normobaric experiments. Freezing was carried out via slow conventional cryopreservation. Different results might be obtained by using vitrification to freeze biological material at a higher pressure.

It is shown that the lower intensity of 2,7-dichlorodihydrofluorescein oxidation processes in inactivated neutrophils exposed to hypomagnetic field (the residual static magnetic field 20 nT) is not related to calcium-mediated mechanisms as shown by the absence of the effect of cell-permeant Ca 2+ chelators, such as 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), on the intensity of the process. This decrease is hardly likely to be caused by effects of a hypomagnetic field on phosphorylation of NADPH-oxidase components, because addition of the protein kinase C inhibitor Ro 316233 decreases the fluorescence intensity of intracellular dichlorodihydrofluorescein little, if at all. Addition of phospholipase C inhibitor U73122 causes a negligible decrease in ROS production in the control and experiment, almost equally. Different concentrations of apocynin increase ROS production in nonactivated neutrophils and this effect is approximately two times lower under hypomagnetic conditions. The decrease in ROS production is more pronounced in cells treated with a hypomagnetic field with the presence of rotenone, indicating that the mitochondrial electron-transport chain is involved in the mechanism of the effect of hypomagnetism.

It has been shown that the decrease in intensity of 2,7-dichlorodihydrofluorescein oxidation processes in inactivated neutrophils under hypomagnetic field exposure (the residual static magnetic field of 20 nT) is not related to the calcium-mediated mechanisms as evidenced by the absence of the effect of cell-permeant Ca2+ chelators such as 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester on the intensity of the process alluded. This decrease is hardly caused by effects of exposure to hypomagnetic field on phosphorylation of NADPH-oxidase components because addition of a protein kinase C inhibitor, Ro 31-6233, did not virtually decrease the fluorescence intensity of intracellular dichlorodihydrofluorescein. Addition of a phospholipase C inhibitor, U73122, led to a negligible decrease in ROS production in control (almost same extent as in the experiment). In the presence of apocynin at different concentrations ROS production in inactivated neutrophils increased and it was 2 times lower under hypomagnetic field conditions. A decrease in ROS production which was more apparent during the experiment after addition of rotenone is indicative of the fact that the mitochondrial electron-transport chain is involved in the mechanism of the effect of a “zero” magnetic field.