Role of Liquid-Liquid Phase Transitions in Mechanism of Erythrocyte Protection During Cooling with CRIHBT-115 Cryopreservative Agent


  • Oleksiy T. Khodko Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv



liquid-liquid phase transitions, crystallization, vitrification, dispersed systems, cryopreservation, cryoinjury, cryoprotectants, erythrocytes, polarized light microscopy


The presence in the system of critical liquid-liquid phase transition (PT) by the mechanism, resulting in formation of dispersion system, namely high-concentrated emulsion, has been established here during cooling when using polarized light microscopy and fixation of critical opalescence phenomenon in erythrocyte concentrate with glycerol-containing cryopreservative agent, designed at the Central Research Institute of Haematology and Blood Transfusion (Russia) (CRIHBT-115 ). The studied cryobiological system displayed no signs of crystallization. A phase behaviour of cryopreservative and supernatant has been studied during cooling-warming cycle. Changes in the volume of cryopreservative and erythrocyte concentrate were comparatively and qualitatively evaluated during cooling. The mechanism of protective action of cryopreservation solution has been determined. The similarity between physical and chemical processes during cooling-warming of erythrocyte cytoplasm and garlic meristem cells (germinal plant tissue) when entering cold anabiosis has been established.


Probl Cryobiol Cryomed 2021; 31(3): 236–248

Author Biography

Oleksiy T. Khodko , Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv

Laboratory of Cryoprotectants


Alberti S, Gladfelter A, Mittang T. Consideration and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell. 2019; 176 (24): 419-34


Anisimov MA. Critical phenomenons in liquids and liquid crystals. Philadelphia, USA: Gordon & Breach Science Publishers: 1991. 431 p.

Bachler J, Handle PH, Giovambattista N, Loerting T. Glass polymorphism and liquid-liquid phase transition in aqueous solutions: experiments and computer simulations. Phys Chem Chem Phys. 2019; 21 (42): 23238-68.


Berestneva ZYa, Kargin VA. [On the mechanism of formation of colloidal particles]. Uspekhi Khimii. 1955; (3): 253-9. Russian.

Bogolyubov DS. [Membrane-less organelles of the eukaryotic cell: basic concepts and principles of formation]. Tsitologiia. 2019; 63(9): 683-703. Russian.

Brangwynne CP. Phase transitions and size scaling of membrane-less organelles. J Cell Biol. 2013; 203: 875-81.


Brazhkin VV. [Can glass-forming liquids be "simple?"]. Uspekhi Fizicheskikh Nauk 2019; 189; (6): 665-72. Russian.


Chechko VYe, Gotsulskyi VYa. [Qualitative analysis of clustering in aqueous alcohol solutions]. Ukrainian Journal of Physics. 2018; 63; (6): 520-5. Ukrainian.


Crowe CD, Keating CD. Liquid-liquid phase separation in artificial cells. Interface Focus [Internet]. 2018 [Cited 2019 Jan 8]; 8:20180032. Available from: https://royalsocietypublishing. org/doi/pdf/10.1098/rsfs.2018.0032.


Cuevas-Velazquez CL, Dinnely JR. Organization out of disorder: liquid-liquid phase separation in plants. Curr Opin Plant Biol. 2018; 45: 68-74.


Davey RJ, Schroeder SLM, ter Horst JH. Nucleation of organic crystals - A molecular perspective. Angew Chem Int Ed. 2013; 52: 2166-79. DOI: 10.1002/anie. 201204824.


Fabelinskii IL. [Molecular dissipation of light]. Moscow: Nauka; 1965. 512 p. Russian.

Feig JSG, Eisenberg DP, Rabin Y. Polarized scanning cryomacroscopy, part 1: Experimental apparatus and observations of vitrification, crystallization, and photoelasticity effects. Cryobiology. 2016; 73: 261-71.


Fuller BJ, Lane N, Benson EE, editors. Life in the frozen state. Boca Raton, USA: CRC Press, 2004. 663p.


Gelfman MI, Kovalevich OV, Yustratov VP, at al. [Colloid chemistry]. St. Petersburg: Lan'; 2004. 336 p. Russian.

Goltsev AN, Grischenko VI, Novak VL, at al. [Cryopreservation of donor blood cells and their long-time storage at low temperature banks]. (Guidelines). Kharkiv: 2016. 33 p. Ukrainian.

Gorelik SS, Dashevsky MYa. [Materials science of semiconductors and dielectrics]. Moscow: Nauka; 2003. 404 p. Russian.

Holmberg K, Jönsson B, Kronberg B, Lindman B. Surfactants and polymers in aqueous solutions. Chichester: John Wiley & Sons, 2002. 562p.


Hyman AA, Weber CA, Jülicher F. Liquid-liquid phase separation in biology. Annu Rev Cell Dev Biol. 2014; 30: 39-58.


Keating CD. Aqueous phase separation as a possible route to compartmentalization of biological molecules. Acc Chem Res 2012; 45: 2114-24.


Kessler UM, Petrenko VE, Liaschenko AK. [Water: structure, condition, solvation] Moscow: Nauka; 2003. 404 p. Russian.

Khodko AT. [Liquid-liquid phase transition as a cause of erythrocyte hemolysis during cooling]. In: Rubin AB, Riznichenko GYu, Anashkina AA, editors. The materials of the VI Congress of Biophysicists of Russia; September 16- 21, 2019, Sochi, Russia. Krasnodar; 2019; Vol. 2; p. 82-3.

Khodko AT, Lysak YuS. Physical and chemical processes in embryonic plant tissue during the transition to the state of cold anabiosis and storage at liquid nitrogen temperature. Low Temperature Physics. 2017; 43: 1196-200.


Koposov GP, Tyagunin AV. [Calorimetric studies of quasi-liquid layer on the surfaces of ice granules]. JETP letters. 2011; (5): 406-9. Russian.

Lee CF, Brangwynne CP, Gharakhani J, et al. Spatial organization of the cell cytoplasm by position-dependent phase separation. Phys Rev Lett. [Internet]. 2013 [Cited 2019 Jan 8]; 111:088101. Available from: pdf/10.1103/PhysRevLett.111.088101.


Ma S. Modern theory of critical phenomena. Westview Press, 2000. 558 p.

Mendeleyev DI. [Solutions]. Moscow: Publishing House of USSR Academy of Sciences; 1959. 1163 p. Russian.

Mollison PL, Sloviter HA. Successful transfusion of previously frozen human red cells. Lancet. 1951; 261 (11): 862-4.


Nekoz IA, Gordiyenko YeA, Kuleshova LG, et al. [Features of hemolysis of erythrocytes in aqueous solutions of glycerol]. In: Pushkar NS, editor. [Cryopreservation of cells and tissues]. Kharkiv: IPCC AS of UkSSR; 1989. p. 54-61. Russian

Papkov SP. [The phase equilibrium in polymer-solvent system]. Moscow: Khimiia; 1981. 272 p. Russian.

Patashinskiy AZ, Pokrovskiy VL. [Fluctuation theory of phase transitions]. Moscow: Nauka; 1982. 382 p. Russian.

Prokhorov GG, Belyayev AM, Prokhorov DG [The basis of clinical cryomedicine]. St. Peterburg-Мoscow: Kniga Po Trebovaniyu; 2017. 608 с. Russian.

Ryzhov VN, Tareyeva EE, Fomin YuD, Tsiok EN. [Complex phase diagrams of systems with isotropic potentials: results of computer simulations]. Uspekhi Fizicheskikh Nauk. 2020; 190(5): 449-73. Russian.


Sarkisov GN. [Structural models of water]. Uspekhi Fizicheskikh Nauk 2006; 176(8): 833-45. Russian.


Style RW, Sai T, Fanelli N, et al. Liquid-liquid phase separation in an elastic network. Phys Rev X [Internet]. 2018 [Cited 2019 Jan 8]; 8(1):011028. Available from: https://journals.


Tropin TV, Schmelzer JWP, Aksenov VL. Modern aspects of the kinetic theory of glass transition. Phys Usp. 2016; 59: 42-66.


Vainshtein BK, Chernov AA, Shuvalov LF, editors. [The modern crystallography]. Moscow: Nauka; 1979. 4 vol. Russian.

Veesler S, Revalor E, Bottini O, Hoff C. Crystallization in the presence of liquid-liquid phase separation. Org Process Res Dev. 2006; 10: 841-5.


Vinograd-Finkel FR, Razumova LL, Kudryashova SN. [The use of radiography to study frozen blood]. Biofizika. 1960; 5(2): 229-34. Russian.

West AR. [Solid state chemistry. Theory and applications]. Part 2. Moscow: Mir; 1988. 336 p. Russian.

Wowk B. Thermodynamic aspects of vitrification. Cryobiology. 2010; 60 (5): 11-22.


Zasimchuk VI, Zasimchuk YeE, Gatsenko AS. [Self-organisation in viscous liquids]. Metallofizika I Noveishie Tekhnologii. 2017; (10): 1435-43. Russian.


Zhegunov GF, Nardid OA, Stegniy BT. [The basis of cryobiology and cryomedicine]. Kharkiv: 2019. 616 p. Russian.




How to Cite

Khodko , O. (2021). Role of Liquid-Liquid Phase Transitions in Mechanism of Erythrocyte Protection During Cooling with CRIHBT-115 Cryopreservative Agent. Problems of Cryobiology and Cryomedicine, 31(3), 236–248.



Theoretical and Experimental Cryobiology