Evaluation of apoptosis stages and posphatidylserine distribution in membrane of cord and peripheral blood nucleated cells at various cryopreservation protocols

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Abstract

Cryopreservation of cord blood nucleated cells for subsequent application in clinical practice is the only way of their long-term storage. The aim of this study was evaluation of the apoptosis stages and phosphatidylserine distribution in the nucleated cells membrane after cryopreservation by different methods. Nucleated cell fractions were frozen under the protection of the cryoprotectants with different mechanism of action. It was shown that nucleated cells isolation in polyglucin and subsequent freezing under 5% DMSO protection, as well as cells isolation by two-step centrifugation method and subsequent freezing under 10% of polyethyleneoxide (PEO) protection, allowed to keep intact most of the cord and peripheral blood cells. Cryopreservation of nucleated cells isolated using Ficoll, regardless of the used cryoprotectant, leads to significant disruption of the lipids asymmetric distribution in membrane and significantly reduces the number of living cells. It has been found that cord blood nucleated cells more resistant to damaging factors of cryopreservation than peripheral blood cells, that is shown in significant differences between the number of live cells in quite all cases before and after cryopreservation .

About the authors

L. A Babijchuk

Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Harkov, Ukraine

O. O Mykhailova

Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Harkov, Ukraine

P. M Zubov

Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Harkov, Ukraine

V. V Ryazantsev

Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Harkov, Ukraine

References

  1. Broxmeyer H.E., Douglas G.W., Hangoc G. et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem. PNAS USA 1989; 86: 3828-32.
  2. Gluckman E., Broxmeyer H.E. 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: 1174-8.
  3. Siena S., Bregni M., Brando B. et al. Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1989; 74: 1905-14.
  4. Бабийчук Л. А., Грищенко В. И., Рязанцев В. В. и др. Новые подходы к проблеме криоконсервирования гемопоэтических клеток пуповинной крови человека. Укр. журнал гематол. i трансфузюл. 2005; 4(д): 122-3.
  5. Сведенцов Е.П., Туманова Т.В., Зайцева О.О. и др. Разработка нового метода сохранения жизнеспособных лейкоцитов в условиях околонулевых температур. Казанский мед. журнал 2008; 4: 558-60.
  6. Hubl W., Iturraspe J., Martinez G.A. et al. Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes. Cytometry 1998; 34: 121-7.
  7. Абдулкадыров К.М., Романенко Н.А., Селиванов Е.А. Наш опыт по заготовке, тестированию и хранению гемопоэтических клеток пуповинной крови. Клеточная трансплантология и тканевая инженерия 2006; 3(1): 63-5.
  8. 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(7): 2207-16.
  9. Михайлова О.А., Бабийчук Л. А., Рязанцев В. В. и др. Оценка жизнеспособности и степени нарушения асимметрии мембран ядросодержащих клеток при различных методах их выделения из цельной пуповинной и донорской крови. Вюник проблем бюлогп i медицини 2011; 4(90): 118-22.
  10. Бабмчук Л.О., Грищенко В.1., Рязанцев В.В. та Ы. Пат. 23499 УкраТна, 012N5/00. Споаб видтення ядровмюних кштин пуповинноТ кровi / заявник и патентовласник 1ПЮК НАН УкраТни. Заявл. 2007.01.22.
  11. Boyum A. Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyte aggregating agents. Scand. J. Clin. Lab. Invest. Suppl. 1968; 97: 31-50.
  12. Бабийчук Л.А., Рязанцев В.В., Зубов П.М. и др. Безотмы-вочный метод криоконсервирования цельной пуповинной крови. Новое в гематологии и трансфузиологии. Межд. научно-практич. рецензир. сб. 2007; 60-3.
  13. Бабмчук Л.О., Грищенко В.1., Гурта Т.М. та Ы. Пат. 92227 УкраТна, А01М/02. Споаб крюконсервування ядровмюних кштин пуповинноТ кровь у тому чи^ стовбурових гемопоетичних кштин / заявник и патентовласник 1ПЮК НАН УкраТни. Заявл. 2008.12.05.
  14. Davis J.M., editor. Basic Cell Culture. A Practical Approach. Oxford: Oxford University Press; 2002.
  15. Abrahamsen J.F., Bakken A.M., Bruserud O. et al. Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Bone Marrow Transplant. 2002; 29(2): 165-71.
  16. Philpott N.J., Turner A.J., Scopes J. et al. The use of 7-aminoactinomycin D identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques. Blood 1996; 87(6): 2244-51.
  17. Koopman G., Reutelingsperger C.P., Kuijten G.A. et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84(5): 1415-20.

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