Cryopreservation of Cord Blood Nucleated Cells Using Non-Penetrating Cryoprotectant PEO-1500

Lyubov A. Babijchuk, Olga A. Mykhailova, Vladimir V. Ryazantsev, Pavel M. Zubov, Regina K. Migunova


Development of wash-free cryopreservation methods for cord blood nucleated cell fraction, including the population of hematopoietic stem/progenitor cells, is an actual task for their long-term storage. Present investigation involved the technique of cord blood nucleated cells cryopreservation, comprising the isolation of cell population by the original two-step centrifugation method, the treatment with non-penetrating cryoprotectant polyethylene oxide with molecular weight of 1500 (PEO-1500) and freezing by the two-step program. This technique enabled to preserve more than 72% CD45+ and 75% CD34+ cells with the viability indices of correspondingly 83 and 91% and higher. Herewith, an increased content of reactive oxygen species (42.7 ± 6.3%) on the background of high cell number and their viability may be considered as a cells’ physiological response to the effect of cryopreservation factors.

Probl Cryobiol Cryomed 2016; 26(1):24-34.


hematopoietic stem/progenitor cells; nucleated cells; cord blood; cryopreservation; non-penetrating cryoprotectant; PEO-1500

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Ademokun I.A., Champan C., Dunn J et al. Umbilical cord blood collection and separation for haemapoietic progenitor cell banking. Bone Marrow Transplant 1997; 19(10): 1023–1028. CrossRef PubMed

Babijchuk L.A., Grischenko V.I., Ryazantsev V.V. et al. New approaches to cryopreservation of human cord blood hematopoietic cells. Ukrainian Journal of Hematology and Transfusiology 2005; 4(d): 122–123.

Babijchuk L.A., Mikhailova O.A., Zubov P.M. et al. Assessment of different populations state of cord and donor blood nucleated cells depending on the cryopreservation method. Problems of Cryobiology 2011; 21(4): 385–394.

Babijchuk L.A., Mykhailova O.O., Zubov P.M. et al. Evaluation of apoptosis stages and posphatidyl serine distribution in membrane of nucleated cells of cord and donor blood under various cryopreservation protocols. Cellular Transplantology and Tissue Engineering 2013; 3(4): 50–54.

Babijchuk L.O., Grischenko V.I., Gurina T.M. et al., inventors. Cryopreservation method for the cord blood nucleated cells including hematopoietic stem cells. Patent of Ukraine 92227. 2010 Oct11.

Babijchuk L.O., Grischenko V.I., Ryazantsev V.V. et al., inventors. Way of isolation of cord blood nucleated cells. Patent of Ukraine 23499. 2007 May25.

Babijchuk L.O., Grischenko V.I., Gurina T.M. et al. Way of cryopreservation of the whole cord blood. Patent of Ukraine 80062. 2007 Aug10.

Bass D.A., Parce J.W., Dechatelet L.R. et al. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 1983; 130(4): 1910–1917. PubMed

Boldyrev A.A. The role of free radicals in functional activity of neurons. In: Dambinova S.A., Arutyunyan A.V., editors. Successes in functional neurochemistry. St. Petersburg: St. Petersburg State University; 2003. p. 301–317.

Broxmeyer H. E., Hangoc G., Cooper S. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992; 89: 4109–4113. CrossRef PubMed

Burgio G.R., Locatelli F. Transplant of bone marrow and cord blood hematopoietic stem cells in pediatric practice, revisited according to the fundamental principles of bioethics. Bone Marrow Transplant 1997; 19(12): 1163–1168. CrossRef PubMed

Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol Today 1994; 15(1): 7–10. CrossRef

Chuikov V.A. The mechanism of cryoprotective efficiency and pharmacological properties of dimethyl sulfoxide. Kriobiologiya 1989; 1: 3–10.

Denning-Kendall P., Donaldson C., Nicol A. et al. Optimal processing of human umbilical cord blood for clinical banking. Exp Hematol 1996; 24(12): 1394–1401. PubMed

Donaldson C., Armitage W.J., Denning-Kendall P.A. et al. Optimal cryopreservation of human umbilical cord blood. Bone Marrow Transplant 1996; 8(4): 725–731.

Dubinina E.E. Role of reactive oxygen species as signaling molecules in the metabolism of tissues under conditions of oxidative stress. Voprosy Meditsinskoy Khimii 2001; 6: 561–581.

Fadok V.A., Voelker D.R., Campbell P.A. et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992; 148: 2207–2216. PubMed

Gluckman E. Hematopoietic stemicell transplants using umbilical-cord blood. N Engl J Med 2001; 344(24): 1860–1861. CrossRef PubMed

Gluckman E. Umbilical cord blood transplant in human. Hematol Cell Ther 1996; 38(5): 393–397. CrossRef PubMed

Gluckman E., Broxmeyer H.A., Auerbach A.D. et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989; 321(17): 1174–1178. CrossRef PubMed

Goltsev A.N., Kalinichenko T.A. Human umbilical cord blood as a source of hematopoietic cells for clinical application. Part 2. Immunological characteristics. Problems of Cryobiology 1998; 2: 3–21.

Ryazantsev V.V., Babijchuk L.A., Mykhailova O.O. et al. The generation of reactive oxygen species by cord blood nucleated cells during cryopreservation. Biotechnologia Acta 2014; 7(4): 100–106. CrossRef

Skulachev V.P. Phenomena of programmed death. Mitochond-ria, cells and organs: the role of reactive oxygen species. Soros Educational Journal 2001; 7(6): 4–10.

Stiff P.J., Koester A.R., Weidner M.K. et al. Autologous bone marrow transplantation using unfractionated cells cryopre-served in dymethylsulfoxide and hydroxyethyl starch without controlled-rate freezing. Blood 1987; 70(4): 974–978. PubMed

Stoian I, Oros A, Moldoveanu E. Apoptosis and free radicals. Biochem Mol Med 1996; 59(2): 93–97. CrossRef PubMed

Tsutsaeva A.A., Tsyganenko A.Y., Zheltyakova I.A. et al. Influence of hypothermic storage before and after cryopreservation on properties of nucleated components and whole cord blood plasma. Problems of Cryobiology 2006; 16(1): 45–55.

Zenkov N.K., Lankin V.Z., Menshikova E.B. Oxidative stress. The biochemical and pathophysiological aspects. Moscow: Nauka/Interperiodica; 2001. PubMed



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