Problems of Cryobiology and Cryomedicine

Modeling cell osmotic behavior during cooling and water crystallization

Tue, 14 Jul 2026 00:00:00 +0200

Selection of cryopreservation parameters, such as cooling rates, is crucial for the successful preservation of cell suspensions. The objective of this study was to assess the impact of cooling rates and nucleation on the survival of testicular interstitial cells (ICs) and to model cell osmotic behavior based on the Kedem-Katchalsky formalism during water crystallization. The ICs were cooled in the serum-free medium supplemented with 0.7 M DMSO and 100 mg/ml dextran 40 (0.7DMSO+D40). The modeling of IC osmotic behavior in the medium, based on experimentally determined water and DMSO membrane permeability coefficients and activation energies, revealed the potential to increase the cooling rate from the "standard" 1 °C/min. This possibility was tested experimentally using higher cooling rates of 2, 5, and 10 °C/min. A cooling rate of 2 °C/min, combined with controlled nucleation parameters, was shown to be beneficial for preserving ICs in 0.7DMSO + D40. The approach including modeling, the use of defined serum-free compositions, controlled nucleation and optimization of cooling rates, can be applied to other cell types cryopreserved in multicomponent cryopreservation media.

Probl Cryobiol Cryomed. 2026; 36(2): 79—88

The effect of prolonged cold exposure on leukocyte indices of dairy cows

Roman V. Mylostyvyi — Tue, 14 Jul 2026 00:00:00 +0200

The effect of prolonged winter cold exposure on leukocyte composition and integral leukocyte indices (ILIs) in Brown Swiss dairy cows was investigated. Cows in the cold stress group were exposed to low ambient temperatures for 10 days (mean –9.6 ± 2.1 °C, minimum –14 °C). Prolonged cold exposure was associated with a decrease in the total leukocyte count, an increase in the proportion of monocytes, and no signifi cant changes in other leukocyte subpopulations. Concurrently, decreases in the neutrophil-to-monocyte ratio (NMR), lymphocyte-to-monocyte ratio (LMR), and index of immune reactivity (IIR) indicated a shift in immune balance towards the monocytic component and reduced immune reactivity. Changes in leukocyte indices refl ected a restructuring of immune homeostasis and activation of nonspecifi c defence mechanisms under low-temperature exposure. The results demonstrate the applicability of leukocyte indices for assessing adaptive changes in dairy cows under prolonged cold exposure.

Probl Cryobiol Cryomed. 2026; 36(2): 89—94

Distribution by transmembrane potential of L929 cells in suspension before and after freezing

Tue, 14 Jul 2026 00:00:00 +0200

Monitoring of cell membrane potential is an important tool for assessing their state during functioning, as well as under the influence of external factors. The distribution of transmembrane potential of L929 cells in cultured suspension and after freezing is determined in this work. It is shown that in the control suspension, cells are in different phases of the cell cycle: a larger fraction of cells (82%) is in a hyperpolarized state of varying degrees (G1 and S-phases), and only a small percentage of them (~7%) are in the division stage and have a low transmembrane potential value (depolarized cells). Freezing the L929 cell suspension in the standard mode (10% DMSO, cooling rate of 1 °C/min to –80 °C with subsequent immersion in liquid nitrogen) practically does not change the part of cells in the hyperpolarized state. At the same time, the fraction of cells in the depolarized state disappears. Obviously, cells that were in the division stage are more sensitive to cryodamage factors. Freezing a suspension of L929 cells with a reduced concentration of DMSO (5%) resulted in a signifi cant decrease in the part of cells in a hyperpolarized state and a signifi cant decrease in the ability of cells to adhere.

Probl Cryobiol Cryomed. 2026; 36(2): 95—105

Pre-sowing seed treatment for enhancing frost resistance of winter wheat

Tue, 14 Jul 2026 00:00:00 +0200

The study investigated the effect of treating winter bread wheat seeds with biologically active preparations or exposing them to negative temperatures on the frost resistance level of wheat seedlings. Cattle placenta extracts obtained by cryogenic molecular fractionation and containing a complex of biologically active substances (BAS) were used as preparations. Pre-sowing cooling of seeds was carried out at temperatures of –20, –40, –80, and –196 °C. The results of two-year experiments on determining the frost resistance of winter wheat revealed a clear trend toward an increase in this index after pre-sowing treatment of seeds by priming with BAS from animal placenta. Such treatment had a positive effect on the least frost-resistant of the studied wheat samples, the Solomiia cultivar, which was the weakest in terms of the biometric indices of the original seed seedlings. The mechanism of action of BAS as biological catalysts that contribute to increasing plant stress resistance is discussed. The role of negative temperatures as prerequisites for stratification has been determined. The data presented in the article testify to the effectiveness of the studied methods of pre-sowing treatment of winter wheat seeds regarding its frost resistance.

Probl Cryobiol Cryomed. 2026; 36(2): 106—114

Freezing regimen affects preservation of canine erythrocytes when using dimethyl sulfoxide

Natalia A. Yershova, Tetiana M. Gurina, Olena E. Nipot, Nataliia M. Shpakova — Tue, 14 Jul 2026 00:00:00 +0200

This study examined how dimethyl sulfoxide (DMSO) concentration and different freezing regimens influence the preservation of canine erythrocytes during cryopreservation. The degree of erythrocyte hemolysis was assessed after thawing and at each stage of cryoprotectant removal. Increasing the DMSO concentration from 7.5 to 10% reduced hemolysis immediately after freeze–thawing by more than two-fold. During cryoprotectant removal, the extent of erythrocyte damage did not differ significantly between samples frozen with 7.5 and 10% DMSO. Overall hemolysis after freeze–thawing varied markedly depending on the cooling protocol. The highest level of cell damage (73%) occurred when samples were frozen in nitrogen vapor followed by immersion in liquid nitrogen. Direct immersion of the samples into liquid nitrogen reduced hemolysis to 37%. The most effective protocol involved briefly immersing the bottom of the cryotube in liquid nitrogen prior to complete freezing, resulting in only 31% hemolysis. This approach initiates crystallization at the lower part of the cryotube, substantially reducing the risk of mechanical cell damage during ice structure formation.

Probl Cryobiol Cryomed. 2026; 36(2): 115—121

Seed viability of less spread introduced conifer plants after storage in liquid nitrogen

Emma R. Arapetyan, Maryna O. Shcherbyna, Yurii M. Usatenko — Tue, 14 Jul 2026 00:00:00 +0200

Tolerance of seeds of plants from the family Cupressaceae (Cypress) Chamaecyparis lawsoniana (Lawson's cypress), Cryptomeria japonica (Japanese cypress), Platycladus orientalis (Oriental broadleaf) and Pinaceae (Pine) Larix kaempferi (Kaempfer's larch), Pseudotsuga menziesii (Menzies' pseudotsuga), Tsuga сanadensis (Canadian hemlock), which according to the International Classification are less spread plants, to storage conditions in liquid nitrogen were investigated for the first time. The results of scientific research into the preservation of seeds in liquid nitrogen, as a source of genetic information, confirmed that the investigated seeds of plants introduced in Western Ukraine retained their viability after storage. Soil germination of cryopreserved seeds increased compared to the control variant on 0.7% for C. japonica, 7.4% for L. kaempferi or decreased in T. canadensis by 2%. The growth and development of the control seedlings do not differ from seedlings obtained from cryopreserved seeds.

Probl Cryobiol Cryomed. 2026; 36(2): 122—127

Effect of craniocerebral hypothermia and cell therapy on autonomic nervous system state in rats

Ivan I. Lomakin, Viacheslav V. Mamontov, Olga V. Kudokotseva — Tue, 14 Jul 2026 00:00:00 +0200

This study investigated the eff ects of rhythmic craniocerebral hypothermia (rCCH), administration of cryopreserved nucleated cord blood cells (NCBCs), and their combined application on autonomic regulation of heart rate and arterial blood pressure (BP) parameters in 12 month old spontaneously hypertensive rats (SHRs) with arterial hypertension (AH). A relationship was established between AH and autonomic nervous system imbalance. SHRs demonstrated a shift in heart rate variability (HRV) indices toward enhanced sympathetic activity, which may indicate reduced neurohumoral regulation and signs of overstrain and asthenization within central regulatory structures. On day 7 aft er the combined application of rCCH and NCBCs, the adaptive compensatory capacity of the organism increased signifi cantly. Although these eff ects diminished by day 30, they remained higher compared to the use of each method separately. In addition, unlike the independent application of rCCH or NCBCs, their combined use resulted in a reduction of both systolic and diastolic BP.

Probl Cryobiol Cryomed. 2026; 36(2): 128—135

Chilling and frost tolerances of a luminous Mycena species (Agaricales, Basidiomycota) in Northern Japan

Tamotsu Hoshino, Makoto Hanzaike, Yoko Ando, Minoru Arimoto, Taiga Kasuya — Tue, 14 Jul 2026 00:00:00 +0200

Probl Cryobiol Cryomed. 2026; 36(2): 136—138

Phase transformation and protein-hormonal profile of human cord blood serum at low temperatures

Tue, 14 Jul 2026 00:00:00 +0200

Probl Cryobiol Cryomed. 2026; 36(2): 139—142