Ramazanov V. V., Volovelskaya E. L., Koptelov V. A., Bondarenko V. A.

Properties of Erythrocytes Frozen in Combined Medium with Polyethylenglycol and 1,2-Propanediol

About the author:

Ramazanov V. V., Volovelskaya E. L., Koptelov V. A., Bondarenko V. A.



Type of article:

Scentific article


During freezing of cell suspensions the components of cryopreservative are exposed to concentrating and have both protective and damaging effect on cells. Cryoprotective effect of polymeric non­ penetrating cryoprotectants is associated with their capability to structure water and maintains it in liquid state at low temperatures during freezing (down to –35°C), providing preservation of reparative properties of membranes. Herewith frozen­thawed cells do not lose the capacity to recover its cation balance. Along with the positive effects polymers concentrating during freezing contribute to a significant dehydration of cells and damage of cell membranes. Therefore the problem of approach search of weakening a negative property of polymers during freezing arises. Cryoprotective effect of penetrating cryoprotectants is associated with their penetration into cells and reduction of their dehydration rate, weakening of intensity of environmental hyperconcentrating as well. Moreover reduction of cell dehydration rate may result in probability increase of formation of intracellular ice crystals. Probably negative property of polymeric (non­penetrating) cryoprotectant during freezing of cell suspensions is possible to weaken during introduction of penetrating cryoprotectant into cryopreservative. Inflow of the last into cells may result in reduction of their dehydration rate and, accordingly, weakening of hypertonic stress during freezing, stipulated by concentrating of non­penetrating cryoprotectant. The decrease in intensity of hypertonic stress may weaken the effect of post­hypertonic stress on cells during freeze­thawing and provide preservation of structural and functional properties of cell membranes. Recently there has been used a combined cryopreservative, containing hydroxyethylated starch and dimethyl sulfoxide being effective during freezing of different cells. In addition the mechanisms of protective efficiency of cryopreservatives, including non­penetrating and penetrating cryoprotectants are not revealed. Osmotic, antioxidative and morphological characteristics of erythrocytes frozen in liquid nitrogen (–196єC) in the medium containing PEG­1500 or in combined medium with PEG­1500 and 1,2­PD were studied. During freezing in PEG­1500 containing medium significant rate of erythrocytes damage (65 %), loss by glutathione cells during cryopreservative washing­out and penetration increase of the rest of cells for H+ ions, morphologically presented by spherocytes were noted. At the same time no significant increase of malondialdehyde concentration and valuable change of indices of gluthatione­dependent enzymes activity (gluthatione reductase and gluthatione peroxidase) during freezing in two presented media was found­out. During additional introduction 1,2­PD into medium the damage rate of erythrocytes during freezing significantly decreases (6 %) with keeping the sufficient osmotic, antioxidative and morphological properties of the remained part of cells after cryopreservative washing­out. The findings enable the suggestion of keeping the osmotic and morphological properties of erythrocytes, frozen in combined medium with PEG­1500 and 1,2­PD, provided by penetration of the latter into the cells and weakening of hypertonic stress, stipulated by NaCl and PEG­1500 concentrating during freezing, which provides maintenance of erythrocyte resistance to posthypertonic stress during thawing.


erythrocytes, osmotic, antioxidative and morphological properties, freezing, combined cryopreservative


  • аграненко В. а. замороженная кровь и ее клиническое применение / В. а. аграненко, л. и. Федорова. – М.: Медици­ на, 1983. – 96 с.
  • ашмарин и. п. быстрые методы статистической обработки и планирование экспериментов / и. п. ашмарин, и. п. Васильев, В. а. амбросов. – л.: из–во ленингр. ун–та. 1975. – 76 с.
  • Владимиров Ю. а. перекисное окисление липидов в биологических мембранах / Ю. а. Владимиров, а. и. арчаков. – М.: наука. 1972. – 270 с.
  • Михнович е. п. криоконсервация эритроцитов при –196о с с проникающими и непроникающими веществами / е. п. Михнович, М. а. рождественская, г. т. недельский // «актуальные вопросы криобиологии и криомедицины». – киев : наук. думка. 1974. – с. 161–164.
  • паніна Ю. Є. Фізико–математична теорія гіпотонічного гемолізу еритроцитів людини : автореф. дис. на здобуття наук. ступеня канд. фіз. –мат. наук : спец. 03. 00. 19 «кріобіологія» / Ю. Є. паніна. – Харків, 1999. – 17 с.
  • разыграев а. В. определение глутатион–пероксидазной активности в сыворотке крови человека с использованием пероксида водорода и 5,5’–дитиобис (2–нитробензойной кислоты) / а. В. разыграев, а. В. арутюнян // клиническая лабораторная диагностика. – 2006. – № 6. – с. 13–16.
  • рамазанов В. В. проявление и устранение эффекта «упаковки» в средах с непроникающими и проникающими крио­ протекторами / В. В. рамазанов, В. а. бондаренко // проблемы криобиологии. – 2009. – т. 19, № 3 – с. 312­323.
  • рамазанов В. В. осмотические свойства эритроцитов, замороженных в средах с непроникающими и проникающими криопротекторами / В. В. рамазанов, В. а. бондаренко // проблемы криобиологии. – 2010. – т. 20, № 1 – с. 47­58.
  • рамазанов В. В. Функционирование системы транспорта ионов н+ при модификации мембран эритроцитов в усло­ виях, моделирующих условия замораживания / В. В. рамазанов, р. Ф. забродский, я. Ю. найдюк, В. а. бондаренко // Вісник проблем біології і медицини. – 2010. – Вип. 3 – с. 186­192.
  • Юсупова л. б. о повышении точности определения активности глутатион–редуктазы эритроцитов / л. б. Юсупова // лабораторное дело. – 1989. – № 4. – с. 19–21.
  • Bakhach J. The cryopreservation of composite tissues. Principles and resent advancement on cryopreservation of different type of tissues / J. Bakhach // Organogenesis. – 2009. – Vol. 5, № 3. – P. 119­126.
  • Beutler E. Red cell metabolism. A manual of biochemical methods / E. . Beutler – New York : Grune&Stratton, 1975. – 160 p.
  • Hoffman J. F. On the mechanism and measurement of shape transformations of constant volume of human red blood cells / J. F. Hoffman // Blood Cells. – 1987. – Vol. 12, № 3. – P. 565­588.
  • Levin R. L. The limiting effects of heat and mass transfer on the osmotic behavior of cells during freezing and thawing / R. L. Levin // Cryobiology. – 1982. – Vol. 19, № 6. – P. 669.
  • Mazur P. Survival of frozen–thawed human red cells as a function of the permeation of glycerol and sucrose / P. Mazur, R. H. Miller // Cryobiology. – 1976. – Vol. 13, № 5. – P. 523–536.
  • Pellerin­Mendes C. In vitro study of the protective effect of trehalose and dextran during freezing of human red blood cells in liquid nitrogen / C. Pellerin­Mendes, L. Million, M. Marchand­Arvier // Cryobiology. – 1997. – Vol. 35, № 2. – P. 173–186.
  • Romano L. Characterization of anion transport system in trout red blood cell / L. Romano, H. Passow // Am. J. Physiol. – 1984. – Vol. 246. – P. 330–338.
  • Woolgar A. E. Some combined effects of hypertonic solutions and changes in temperature on post­hypertonic haemolysis of human red blood cells / A. E. Woolgar, C. J. Morris // Cryobiology. – 1973. – Vol. 10. – р. 82–86.

Publication of the article:

«Bulletin of problems biology and medicine» Issue 3 part 2 (111), 2014 year, 230-236 pages, index UDK 577. 352. 4: 612. 111: 547. 42