N. E. Shvirst’s research while affiliated with Russian Academy of Sciences and other places

Background: It is well known that body temperature maintenance between 20 and 35°C prevents hypoxic damage. However, data regarding the ideal duration and permissible temperature boundaries for ultra-deep hypothermia below 20°C are rather fragmentary. The aim of the present study was to determine the time limits of reversible clinical death in rats subjected to ultra-deep hypothermia at 1-8°C. Results: Rat survival rates were directly dependent on the duration of clinical death. If clinical death did not exceed 35 min, animal viability could be restored. Extending the duration of clinical death longer than 45 min led to rat death, and cardiac functioning in these animals was not recovered. The rewarming rate and the lowest temperature of hypothermia experienced did not directly influence survival rates. Conclusions: In a rat model, reversible ultra-deep hypothermia as low as 1-8°C could be achieved without the application of hypercapnia or pharmacological support. The survival of animals was dependent on the duration of clinical death, which should not exceed 35 min.

We studied the effect of xenon on the survival rate of the spermatozoa of the common frog Rana temporaria during slow freezing with saturation of the suspension with xenon at a pressure of up to 1.2 bar. The cryoprotective properties of xenon were analyzed in comparison with nitrogen. No specific cryoprotective effect of xenon was revealed. Viability of spermatozoa pretreated with xenon at atmospheric pressure (0 bar) or under excess pressure of 0.6 bar and frozen in a cryoprotective medium with dimethylformamide, sucrose, and BSA did not differ significantly. The use of overpressure of xenon of 1.0 or 1.2 bar in the pretreatment and freezing process significantly impaired viability of the biomaterial.

Any biological material contains dissolved gases that affect physical and biological processes associated with cooling and freezing. However, in the cryobiology literature, little attention has been paid to the effect of gasses on cryopreservation. We studied the influence of helium, neon, krypton, xenon, argon, nitrogen, and sulfur hexafluoride on the survivability of HeLa and L929 cell lines during cryopreservation. Saturation of a cell suspension with helium, neon, and sulfur hexafluoride enhanced survival of HeLa and L929 cells after cryopreservation. Helium exerted the most significant effect. For a range of noble gases, the efficiency of the positive effect decreased as the molecular mass of the gas increased. This paper discusses possible mechanisms for the influence of gases on the cryopreservation of biological material. The most probable mechanism is the disruption of the frozen solution structure with gas-filled microbubbles produced during water crystallization. Ultimately, it was concluded that helium and neon can be used to improve methods for cryopreservation of cell suspensions with a low concentration of conventional penetrating cryoprotectants or even without them.

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.

High organoprotective properties of a carbon monoxide (CO)–oxygen (O2) gas mixture were confirmed after prolonged (24-h) preservation of the papillary muscle and an isolated rat heart at 4°C. Hypothermic preservation in the high-pressure gas mixture (6 atm) provided efficient restoration of the contractile activity of the isolated rat heart after 24-h storage at 4°C. The isolated retrograde-perfused Langendorff heart performed physically relevant mechanical work, which was similar in duration to that of an intact control heart. Staining with triphenyltetrazolium chloride did not detect infarcted regions in the myocardium. After preservation, the heart tissue was highly capable of performing its function in a test for electrically stimulated contractile activity of papillary muscles. In the test group, The frequency–intensity relationship, the potentiation effect induced by a pause, and the response to stimulation with isoproterenol of test hearts generally corresponded to the parameters of a normal rat myocardium. A sheep heart, which is comparable in size and weight to a human heart, was for the first time successfully preserved using the gas mixture. Normal heartbeat was spontaneously restored after the start of perfusion in all experiments. Histology did not detect a significant difference between test and control sheep hearts. The normal tissue structure of the myocardium was preserved in the test hearts. The 24-h preservation achieved in the study was four times longer than the maximum allowable preservation time of standard static cold storage. The results obtained with the large laboratory animal heart model showed that the hypothermic preservation protocol is promising for prolonged storage of human hearts.

One of the possible approaches to a new method of cryopreservation seems to be the controlled formation of a multitude of small crystals in an object, which, due to their size, will not damage cellular structures. Managing the crystal formation, given the stochastic nature of the process, is an extremely difficult task. Theoretically, it is simplified if there is a sufficient number of changeable physical parameters, affecting the process. From this point of view, the use of ice-like gas hydrates for the purposes of cryopreservation seems to be a promising option. We investigated the process of growth of xenon gas hydrates via standard microscopy under different conditions using the specialized optical cell for observation at elevated pressures. The formation of crystals was observed in the system “supercooled liquid–xenon–water vapor” at negative, near-zero and positive values of temperature, and pressure of xenon up to 8 atmospheres. The morphology of xenon hydrate crystals observed in the experiments was analyzed and classified into five categories. The influence of physical conditions on the predominant crystal morphology was also studied. We found no evidence that the possible damaging effect of hydrate crystals should be less severe than of ice crystals.

The effect of helium on cell survival during cryopreservation was studied using the HeLa and L929 cell lines. Cell suspensions were incubated in an atmosphere of helium, nitrogen, or air and frozen in the presence or absence of glycerol as a cryoprotectant. After thawing, the cell viability was evaluated by the Trypan Blue exclusion test and culture development for 18 h. Helium was found to provide better preservation of cell suspensions compared with nitrogen and air. The positive effect of helium was the greatest in the case of freezing without cryoprotectants (the HeLa cell survival increased by a factor of 1.5–2) and somewhat lower in the case of freezing in the presence of low glycerol concentrations (the L929 cell survival increased by a factor of 1.2–1.5 in the presence of 3% glycerol). Use of helium for cell suspensions may improve cryopreservation methods by making it possible to reduce the concentrations of conventional cryoprotectants, which are generally highly toxic and undesirable to use for cryopreservation of biological material for medical applications.

The absorption spectra of liquid water and different aqueous solutions were analyzed in a terahertz frequency domain (from 6 to 200 cm-1) which characterize the collective dynamics of water molecules. The particular attention was paid to the relaxation process in the range of ~6-80 cm-1. The physical essence of this process on the molecular level is still unclear. We found that the amplitude of this relaxation process correlates with the degree of destruction of water structure. The obtained data allowed us to interpret this process as a monomolecular relaxation of free water molecules. Based on a consideration of the water polarization in the electric field we proposed a method of calculation of the amount of free water molecules in solution.

We analyzed spectra of light and heavy water at temperatures from 4 up to 50°C in a frequency range of 0.15 to 6.5 THz. It was shown that the amplitude of high-frequency relaxation absorption band with its maximum at 0.5 THz extends with increasing temperature and this temperature dependence for light water has a marked feature at 35–40°C as a sharp growth. This fact is noteworthy because this range corresponds to physiological values of a body temperature of the warm-blooded organisms. At the same time, the analogous temperature dependence for heavy water in the considered temperature range lacks this particular feature. Thus, the water with its properties differs significantly not only from other fluids, but also from its own isotopologues.