Optimization of Cryopreservation Technique for Human Cord Blood Nucleated Cells Using Combination of Cryoprotectant DMSO and Antioxidant N-acetyl-L-cysteine

Authors

  • Olena E. Makashova Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv
  • Lyubov O. Babijchuk Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv
  • Oksana L. Zubova Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv
  • Pavlo M. Zubov Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv

DOI:

https://doi.org/10.15407/cryo26.04.295

Keywords:

human cord blood, nucleated cells, reactive oxygen species, cryopreservation, dimethyl sulfoxide, antioxidants, N-acetyl-L-cysteine

Abstract

The paper evaluated the efficiency of N-acetyl-L-cysteine (AC) antioxidant for cryopreservation of human cord blood nucleated cells (CBNCs) with various concentrations of endocellular cryoprotectant dimethyl sulfoxide (DMSO). It has been found that rise in DMSO concentration (from 2.5 and 5 up to 7.5% and 10%) and exposure time of the CBNCs suspension with cryoprotectant (from 15 to 30 min and longer) resulted in a significant increase in the amount of cells with excess reactive oxygen species (ROS) (from (7.5 ± 0.8)% at 5% DMSO and 15-min incubation to (28.9 ± 3.2)% with 10% DMSO and 60 min incubation), decrease in their viability and preservation rate. Supplementing 10 mM AC to the cryopreservation medium led to a reduction in the amount of cells with excess ROS and rise of their preservation rate and viability at the stage of equilibration with cryoprotectant, as well as after freeze-thawing of CBNCs suspension. Maximum effect was achieved after AC supplementing to the media with 7.5 and 10% DMSO concentrations. We proved that the use of antioxidant contributed to the rise in preservation rate and viability of CBNCs if cryoprotectant concentration and exposure time with it were optimal.

Probl Cryobiol Cryomed 2016; 26(4): 295–307.

Author Biographies

Olena E. Makashova, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv

Department of Cryocytology

Lyubov O. Babijchuk, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv

Department of Cryocytology

Oksana L. Zubova, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv

Department of Cryocytology

Pavlo M. Zubov, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv

Department of Cryocytology

References

Armitage S. Cord blood banking standards: autologous versus altruistic. Front Med (Lausanne) 2015; 2: 94. CrossRef

Babijchuk L.A., Makashova O.Ye., Zubov P.M., Zubova O.L. Estimation of antioxidant properties of N-acetyl-L-cysteine during cord blood cryopreservation with DMSO. In: Advances in cell biology and biotechnology: Proceedings of the International Conference of Ukrainian Society of Cell Biology; 2015 Oct 11–13; Lviv.

Babijchuk L.A., Makashova O.Ye., Zubova O.L. et al. Using of N-acetyl-L-cysteine antioxidant for increasing in cord blood nucleated cells recovery and viability cryopreserved with DMSO. Proceedings of Conference on Actual Questions of Transfusiology and Clinical Medicine; 2015 Oct 6-7; Kirov. p. 32–35.

BabÑ–jchuk L.O., Grischenko V.І., GurÑ–na T.M. et al., inventors. Cryopreservation method of cord blood nucleated cells including hematopoietic stem cells. Patent of Ukraine â„– 92227, IPC Ð01N1/02. 2010 Oct 11.

Ballen K., Gluckman E., Broxmeyer H.E. Umbilical cord blood transplantation: the first 25 years and beyond. Blood 2013; 122(4): 491–498. CrossRef PubMed

Baust J.G., Gao D., Baust J.M. Cryopreservation: An emerging paradigm change. Organogenesis 2009; 5 (3): 90–96. CrossRef PubMed

Chen G., Yue A., Ruan Z. et al. Comparison of the effects of different cryoprotectants on stem cells from umbilical cord blood. Stem Cells Int 2016; (2016): 1396783–1396790. CrossRef

Chen X., Zhong H. Z, Xu Z. et al. 2',7'-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radic Res 2010; 44(6): 587–604. CrossRef PubMed

Davis J.M., editor. Basic cell culture. A practical approach. Oxford University Press, Oxford; 2002.

Fiorentini C., Falzano L., Rivabene R. et al. N-acetylcysteine protects epithelial cells against the oxidative imbalance due to Clostridium difficile toxins. FEBS Letters 1999; 453(1–2): 124–128. CrossRef

Fry L.J., Querol S., Gomez S.G. et al. Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation. Vox Sang 2015; 109(2): 181–190. CrossRef PubMed

Gluckman E. History of cord blood transplantation. Bone Marrow Transplantation 2009; 44(10): 621–626. CrossRef PubMed

Hayakawa J., Joyal E.G., Gildner J.F. et al. 5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO. Transfusion 2010; 50(10): 2158–2166. CrossRef PubMed

Lafolla M.A., Tay J., Allan D.S. Transplantation of umbilical cord blood-derived cells for novel indications in regenerative therapy or immune modulation: a scoping review of clinical studies. Biol Blood Marrow Transplant 2014; 20(1): 20–25. CrossRef PubMed

Khomenko V.I., Bychkov V.V., Bazyka D.A. State of development of hematopoietic stem cell transplantation in Еurope and world. Lik Sprava 2014; 7–8: 117–121.

Kim K.M., Huh J.Y., Hong S.S., Kang M.S. Assessment of cell viability, early apoptosis, and hematopoietic potential in umbilical cord blood units after storage. Transfusion 2015; 55(8): 2017–2022. CrossRef PubMed

Makashova E.Ye., Zubova O.L., Zubov P.M. Use of N-Acetyl-L-Cysteine antioxidant in cryopreservation of cord blood nucleated cells. Problems of Cryobiology and Cryomedicine 2016; 26(2): 183. CrossRef

Millea P.J. N-acetylcysteine: multiple clinical applications. Am Fam Physician 2009; 80(3): 265–269. PubMed

Mitrus I., Smagur A., Giebel S. et al. A faster reconstitution of hematopoiesis after autologous transplantation of hematopoietic cells cryopreserved in 7.5% dimethyl sulfoxide if compared to 10% dimethyl sulfoxide containing medium. Cryobiology 2013; 67(3): 327–331. CrossRef PubMed

Passweg J.R., Baldomero H., Bader P. et al. Hematopoietic stem cell transplantation in Europe 2014: more than 40 000 transplants annually. Bone Marrow Transplant 2016; 51(6): 786–792. CrossRef PubMed

Ray P.D., Huang B.W., Tsuji Y. Reactive oxygen species (ROS): homeostasis and redox regulation in cellular signaling. Cell Signal 2012; 24(5): 981–990. CrossRef PubMed

Rocha V., Labopin M., Sanz G. et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. En Engl J Med 2004; 351 (722): 2276–2285. CrossRef PubMed

Rushworth G.F., Megson I.L. Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits. Pharmacol Ther 2014; 141(2): 150–159. CrossRef PubMed

Schmid I.W., Krall J., Uittenbogaart C.H. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color laser flow cytometry. Cytometry 1992; 13: 204–208. CrossRef PubMed

Soh N. Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal Bioanal Chem 2006; 386(3): 532–543. CrossRef PubMed

Thannickal V.J., Fanburg B.L. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000; 279(6): 1005–1028. PubMed

Wang L.L., Jin L., Xu H.M., Hao Y.W. Correlation between reactive oxygen species of cryopreserved peripheral blood mononuclear cells and expression of homing adhesion molecules on peripheral blood hematopoietic stem/progenitor cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2012; 20(6): 1452–1456.

Wardman P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 2007; 43(7): 995–1022. CrossRef PubMed

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Published

2016-12-23

How to Cite

Makashova, O. E., Babijchuk, L. O., Zubova, O. L., & Zubov, P. M. (2016). Optimization of Cryopreservation Technique for Human Cord Blood Nucleated Cells Using Combination of Cryoprotectant DMSO and Antioxidant N-acetyl-L-cysteine. Problems of Cryobiology and Cryomedicine, 26(4), 295–307. https://doi.org/10.15407/cryo26.04.295

Issue

Section

Theoretical and Experimental Cryobiology