DMSO-Free Cryopreservation of Human Umbilical Cord Tissue

Human umbilical cord represents a source of multipotent stromal cells of a supreme therapeutic potential. The cells can be isolated from either fresh or cryopreserved umbilical cord tissues. DMSO is a cryoprotectant most commonly used for preservation of umbilical cord tissues; however, cyto- and genotoxicity of this compound is evident and well documented. In the present study we performed successful cryopreservation of the umbilical cord tissue using other cryoprotectants: propylene glycol, ethylene glycol, and glycerol. Of these, 1.5 M ethylene glycol and 20% glycerol turned out to be the best in terms of the preservation of living cells within the frozen tissue, early onset of migration of these cells out of the thawed explants, and overall efficacy of multipotent stromal cell isolation. Cryobanking of tissues can improve availability of multiple cell products for medical purposes and promote the development of personalized medicine.

This is a preview of subscription content, access via your institution.


  1. 1.

    Kostyaev AA, Martusevich AK, Andreev AA. Toxicity of cryoprotectants and cryoconservants on their basis for blood components and bone marrow (review article). Nauch. Obozrenie. Med. Nauki. 2016;(6):54-74. Russian.

  2. 2.

    Romanov YA, Balashova EE, Volgina NE, Kabaeva NV, Dugina TN, Sukhikh GT. Isolation of Multipotent Mesenchymal Stromal Cells from Cryopreserved Human Umbilical Cord Tissue. Bull. Exp. Biol. Med. 2016;160(4):530-534.

    CAS  Article  Google Scholar 

  3. 3.

    Arutyunyan I, Elchaninov A, Makarov A, Fatkhudinov T. Umbilical cord as prospective source for mesenchymal stem cellbased therapy. Stem Cells Int. 2016;2016. ID 6901286. doi:

    Article  Google Scholar 

  4. 4.

    Badowski M, Muise A, Harris DT. Mixed effects of longterm frozen storage on cord tissue stem cells. Cytotherapy. 2014;16(9):1313-1321.

    CAS  Article  Google Scholar 

  5. 5.

    Best BP. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Res. 2015;18(5):422-436.

    Article  Google Scholar 

  6. 6.

    Carnevale G, Pisciotta A, Riccio M, De Biasi S, Gibellini L, Ferrari A, La Sala GB, Bruzzesi G, Cossarizza A, de Pol A. Optimized Cryopreservation and banking of human bone-marrow fragments and stem cells. Biopreserv. Biobank. 2016;14(2):138-148.

    CAS  Article  Google Scholar 

  7. 7.

    Choudhery MS, Badowski M, Muise A, Harris DT. Utility of cryopreserved umbilical cord tissue for regenerative medicine. Curr. Stem Cell Res. Ther. 2013;8(5):370-380.

    CAS  Article  Google Scholar 

  8. 8.

    Choudhery MS, Badowski M, Muise A, Pierce J, Harris DT. Cryopreservation of whole adipose tissue for future use in regenerative medicine. J. Surg. Res. 2014;187(1):24-35.

    CAS  Article  Google Scholar 

  9. 9.

    Da-Croce L, Gambarini-Paiva GH, Angelo PC, Bambirra EA, Cabral AC, Godard AL. Comparison of vitrification and slow cooling for umbilical tissues. Cell Tissue Bank. 2013;14(1):65-76.

    Article  Google Scholar 

  10. 10.

    Ding DC, Chang YH, Shyu WC, Lin SZ. Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy. Cell Transplant. 2015;24(3):339-347.

    Article  Google Scholar 

  11. 11.

    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317.

    CAS  Article  Google Scholar 

  12. 12.

    Fleury A, Pirrello O, Maugard C, Mathelin C, Linck C. Breast cancer and ovarian tissue cryopreservation: Review of the literature. J. Gynecol. Obstet. Hum. Reprod. 2018. May 21. pii: S2468-7847(18)30172-7. doi:

    Article  Google Scholar 

  13. 13.

    Galvao J, Davis B, Tilley M, Normando E, Duchen MR, Cordeiro MF. Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J. 2014;28(3):1317-1330.

    CAS  Article  Google Scholar 

  14. 14.

    Giugliarelli A, Urbanelli L, Ricci M, Paolantoni M, Emiliani C, Saccardi R, Mazzanti B, Lombardini L, Morresi A, Sassi P. Evidence of DMSO-induced protein aggregation in cells. J. Phys. Chem. A. 2016;120(27):5065-5070.

    CAS  Article  Google Scholar 

  15. 15.

    Iftimia N, Ferguson RD, Mujat M, Patel AH, Zhang EZ, Fox W, Rajadhyaksha M. Combined reflectance confocal microscopy/optical coherence tomography imaging for skin burn assessment. Biomed. Opt. Express. 2013;4(5):680-695.

    Article  Google Scholar 

  16. 16.

    Johnson S, Rabinovitch P. Ex vivo imaging of excised tissue using vital dyes and confocal microscopy. Curr. Protoc. Cytom. 2012. Chapter 9:Unit 9.39. doi:

  17. 17.

    Kalaszczynska I, Ferdyn K. Wharton’s jelly derived mesenchymal stem cells: future of regenerative medicine? Recent findings and clinical significance. Biomed. Res. Int. 2015;2015. ID 430847. doi:

    Article  Google Scholar 

  18. 18.

    Moll G, Geißler S, Catar R, Ignatowicz L, Hoogduijn M.J, Strunk D, Bieback K, Ringdén O. Cryopreserved or fresh mesenchymal stromal cells: only a matter of taste or key to unleash the full clinical potential of MSC therapy? Adv. Exp. Med. Biol. 2016;951:77-98.

  19. 19.

    Morris C, de Wreede L, Scholten M, Brand R, van Biezen A, Sureda A, Dickmeiss E, Trneny M, Apperley J, Chiusolo P, van Imhoff GW, Lenhoff S, Martinelli G, Hentrich M, Pabst T, Onida F, Quinn M, Kroger N, de Witte T, Ruutu T; Chronic Malignancies and Lymphoma Working Parties of EBMT. Should the standard dimethyl sulfoxide concentration be reduced? Results of a European Group for Blood and Marrow Transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide. Transfusion. 2014;54(10):2514-2522.

    CAS  Article  Google Scholar 

  20. 20.

    Nakamura Y, Obata R, Okuyama N, Aono N, Hashimoto T, Kyono K. Residual ethylene glycol and dimethyl sulphoxide concentration in human ovarian tissue during warming/thawing steps following cryopreservation. Reprod. Biomed. Online. 2017;35(3):311-313.

    CAS  Article  Google Scholar 

  21. 21.

    Park BW, Jang SJ, Byun JH, Kang YH, Choi MJ, Park WU, Lee WJ, Rho GJ. Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells. J. Tissue Eng. Regen. Med. 2017;11(2):489-500.

    CAS  Article  Google Scholar 

  22. 22.

    Rodríguez L, Velasco B, García J, Martín-Henao GA. Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing. Transfusion. 2005;45(8):1391-1397.

    Article  Google Scholar 

  23. 23.

    Rozati H, Handley T, Jayasena CN. Process and pitfalls of sperm cryopreservation. J. Clin. Med. 2017;6(9). pii. E89. doi:

  24. 24.

    Sharma S, Venkatesan V, Prakhya BM, Bhonde R. Human mesenchymal stem cells as a novel platform for simultaneous evaluation of cytotoxicity and genotoxicity of pharmaceuticals. Mutagenesis. 2015;30(3):391-399.

    CAS  Article  Google Scholar 

  25. 25.

    Shivakumar SB, Bharti D, Subbarao RB, Jang SJ, Park JS, Ullah I, Park JK, Byun JH, Park BW, Rho GJ. DMSO- and serumfree cryopreservation of Wharton’s jelly tissue isolated from human umbilical cord. J. Cell. Biochem. 2016;117(10):2397-2412.

    CAS  Article  Google Scholar 

  26. 26.

    Svalgaard JD, Haastrup EK, Reckzeh K, Holst B, Glovinski PV, Gørløv JS, Hansen MB, Moench KT, Clausen C, Fischer-Nielsen A. Low-molecular-weight carbohydrate Pentaisomaltose may replace dimethyl sulfoxide as a safer cryoprotectant for cryopreservation of peripheral blood stem cells. Transfusion. 2016;56(5):1088-1095.

    CAS  Article  Google Scholar 

  27. 27.

    Unni S, Kasiviswanathan S, D’Souza S, Khavale S, Mukherjee S, Patwardhan S, Bhartiya D. Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant. Fertil. Steril. 2012;97(1):200-8.e1.

    Article  Google Scholar 

  28. 28.

    Yang Y, Melzer C, Bucan V, von der Ohe J, Otte A, Hass R. Conditioned umbilical cord tissue provides a natural threedimensional storage compartment as in vitro stem cell niche for human mesenchymal stroma/stem cells. Stem Cell Res Ther. 2016;7:28. doi:

  29. 29.

    Yuan C, Gao J, Guo J, Bai L, Marshall C, Cai Z, Wang L, Xiao M. Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes. PLoS One. 2014;9(9):e107447. doi:

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Т. Kh. Fatkhudinov.

Additional information

Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 3, pp. 180-188, September, 2018

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Arutyunyan, I.V., Strokova, S.О., Makarov, А.V. et al. DMSO-Free Cryopreservation of Human Umbilical Cord Tissue. Bull Exp Biol Med 166, 155–162 (2018).

Download citation

Key Words

  • tissue cryopreservation
  • umbilical cord
  • cryoprotectant
  • dimethyl sulfoxide
  • multipotent stromal cells