Cell and Organ Transplantology. 2020; 8(1):51-57.
Cryopreservation of human Wharton’s jelly multipotent mesenchymal stromal cells with reduced concentration of dimethyl sulfoxide
Tsymbaliuk V.1,3, Deryabina O.2,4, Shuvalova N.2, Verbovska S.3, Pichkur L.3, Olexenko N.3 , Kordium V.2,4
- 1National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
- 2State Institute of Genetic and Regenerative Medicine of the National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
- 3Romodanov State Institute of Neurosurgery of the National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
- 4Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, Kyiv, Ukraine
The urgent problem of long-term storage of multipotent mesenchymal stromal cells (MMSCs) is to improve the protocol of their cryopreservation for further application maintaining the therapeutic properties and minimizing the risks of adverse effects on the health of the recipient. As a standard cryoprotectant, a mixture of 90 % fetal bovine serum (FBS) and 10 % dimethyl sulfoxide (DMSO) is used, which, however, can cause a variety of adverse reactions. Therefore, it is important to study the possibility of reducing the concentration of potentially dangerous DMSO by adding other components to the mixture for cell cryopreservation.
Purpose. To determine the efficiency of cryopreservation of human Wharton’s jelly MMSCs using cryoprotectants of different composition by studying the proliferative activity, phenotype and features of cell morphology in culture in vitro.
Materials and methods. The cryoprotective effect of various combinations of DMSO, ethylene glycol, sucrose and trehalose was studied. The efficacy was assessed by cell viability, their adhesive properties, expansion rate and monolayer formation, as well as the expression of main MMSCs markers.
Results. It is shown that the most effective combination is 4 % DMSO with 6 % trehalose which provides the highest level of preservation of cell viability, as well as their adhesive and proliferative properties during thawing. Other combinations of the cryoprotectant components showed a much slower cell division, in some cases, the monolayer was not formed at all. For all investigated variants, the main surface markers of MMSCs were preserved.
Conclusions. The obtained results indicate the possibility of reducing the concentration of DMSO to 4 % in the freezing medium for MMSCs cryopreservation while maintaining their viability, proliferative activity and common surface markers.
Key words: multipotent mesenchymal stromal cells; Wharton’s jelly; cell cryopreservation; DMSO; trehalose; sucrose, ethylene glycol
Full Text PDF (eng) Full Text PDF (ua)
|1. Pichkur LD, Verbovs’ka SA, Akinola ST, Chitaeva GE. Basic pathogenetic mechanisms of demyelination in the central nervous system and its correction limitations. Ukrainian Neurological Journal. 2017; 2:12-19. http://www.ukrneuroj.com.ua/svizhij_nomer.php?nid=43.|
|2. Maslova O, Novak M, Kruzliak P. Umbilical cord tissue-derived cells as therapeutic agents. Stem Cells International. 2015; 2015:1-10. DOI:10.1155/2015/150609.
|3. Vangsness CT, Sternberg H, Harris L. Umbilical cord tissue offers the greatest number of harvestable mesenchymal stem cells for research and clinical application: a literature review of different harvest sites. Arthroscopy. 2015; 31(9):1836-1843. DOI:10.1016/j.arthro.2015.03.014.
|4. Troyer DL, Weiss M L. Concise review: Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells. 2008; 26(3):591-599. DOI:10.1634/stemcells.2007-0439.
|5. Fong CY, Chak LL, Biswas A, Tan JH, Gauthaman K, Chan WK, et al. Human Wharton’s jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells. Stem Cell Rev. 2011; 7:1-16. DOI:10.1007/s12015-010-9166-x
|6. Vaslovych VV, Pichkur LD, Malysheva TA, Akinola ST, Verbovska SA, Toporova OK, et al. Ultrastructural changes in spinal cord of rats with experimental allergic encephalomyelitis in the background of mesenchymal stem cells and interleukin-10. J Clin Exp Med Res, 2018; 1:17-30. http://essuir.sumdu.edu.ua/handle/123456789/67929.|
|7. Pichkur LD, Semenova VM., Verbovska SA, Oleksenko NP, Akinola ST. The features of experimental allergic encephalomyelitis after stem cells transplantation. Ukrainian Neurosurgical Journal. 2017; 2:27-33. DOI: 10.25305/unj.104500.
|8. Cohen JA. Mesenchymal stem cell transplantation in multiple sclerosis. J Neurol Sci. 2013; 333(1-2):43-9. DOI:10.1016/j.jns.2012.12.009.
|9. Guo AC, Chu T, Liu XQ, Su HX, Wu WT. Reactivated astrocytes as a possible source of oligodendrocyte precursors for remyelination in remitting phase of experimental autoimmune encephalomyelitis rats. Am J Transl Res. 2016; 8(12):5637-45.|
|10. Best BP. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Res. 2015; 18(5):422-36. DOI:10.1089/rej.2014.1656.
|11. Crowley JR, Rene A, Valery RC. The recovery, structure and function of hyman blood leukocytes after freeze-preservation. Cryobiology. 1974; 11(3):395-409. http://doi.org/10.1016/0011-2240(74)90106-0.
|12. Siddiqui M, Parvin R, Giasuddin M, Chowdhury S, Islam M, Chowdhury E. The effect of different concentrations of Dimethyl sulfoxide (DMSO) and glycerol as cryoprotectant in preserving Vero cells. Bangladesh Veterinarian. 2017; 33(1):1-7. https://doi.org/10.3329/bvet.v33i1.33307.
|13. Bersenev AV. Convulsions and coma as complications of DMSO-associated toxicity during infusion of hematopoietic cells in bone marrow transplantation. Cell Transplantology and Tissue Engineering. 2006; 1:31-32.|
|14. Balci D, Can A. The assessment of cryopreservation conditions for human umbilical cord stroma-derived mesenchymal stem cells towards a potential use for stem cell banking. Curr. Stem Cell Rep. 2013; 8(1):60-72. DOI:10.2174/1574888×11308010008.
|15. Asghar W, El Assal R, Shafiee H, Anchan RM, Demirci U. Preserving human cells for regenerative, reproductive, and transfusion medicine. Biotechnol J. 2014; 9(7):895-903. DOI:10.1002/biot.201300074.
|16. Mantri S, Kanungo S, Mohapatra P. Cryoprotective Effect of Disaccharides on Cord Blood Stem Cells with Minimal Use of DMSO. Indian Journal of Hematology and Blood Transfusion. 2014; 31(2):206-212. DOI:10.1007/s12288-014-0352-x.
|17. Yong KW, WanSafwani WK, Xu F, WanAbas WA, Choi JR, Pingguan-Murphy B. Cryopreservation of human mesenchymal stem cells for clinical applications: current methods and challenges. Biopreservation and biobanking. 2015; 13(4):231-9. DOI:10.1089/bio.2014.0104. PMID: 26280501.
|18. Tsymbaliuk, Pichkur LD, Deryabina OG, Maslova OA, Shuvalova NS, Verbovs’ka SA, et al. Phenotypic characteristics and proliferative potential of human mesenchymal stem cells from Wharton jelly during cultivation ex vivo. In: Aspects of cell cultivation methods in neurobiology and neurooncology. Edited by Semenova VM. Kyiv: Interservice, 2018. p. 295-304.|
|19. Roth V. Doubling Time Calculator. 2006. http://www.doubling-time.com/compute.php.|
|20. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8(4):315-7. DOI: 10.1080/14653240600855905.
|21. Mohammadi Z, Tavakkol Afshari J, Keramati MR, Hamidi Alamdari D, Ganjibakhsh M, Moradi Zarmehri A, et al. Differentiation of adipocytes and osteocytes from human adipose and placental mesenchymal stem cells. Iran J Basic Med Sci 2015; 18:259-266.|
|22. Acharya C, Adesida A, Zajac P, Mumme M, Riesle J, Martin I, et al. Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation. J Cell Physiol. 2012; 227(1):88-97. DOI: 10.1002/jcp.22706.
|23. Wosnitza M, Hemmrich K, Groger A, Graber S, Pallua N. Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation. Differentiation. 2007; 75:12-23.
|24. Kim BS, Kim JS, Chung YS, Sin YW, Ryu KH, Lee J, et al. Growth and osteogenic differentiation of alveolar human bone marrow-derived mesenchymal stem cells on chitosan/hydroxyapatite composite fabric. J Biomed Mater Res A. 2013; 101:1550-1558.
|25. Gimble JM, Guilak F, Nuttall ME, Sathishkumar S. In vitro Differentiation Potential of Mesenchymal Stem Cells. Transfus Med Hemother. 2008; 35:228-238.
|26. Kruglyakov PV, Polyntsev DG, Vyide SK, Kislyakova TV. Sreda dlya kriokonservirovaniya mezenkhimal’nykh stvolovykh kletok i biotransplantat s ee ispol’zovaniem [Medium for cryopreservation of mesenchymal stem cells and biograft with its use]. Opisanie k Pat. RF – Description to Pat. RU2303631C1, pub. 19.05.2006. [In Russian]|
|27. Matsumura K, Hayashi F, Nagashima T, Hyon SH. Long-term cryopreservation of human mesenchymal stem cells using carboxylated poly-l-lysine without the addition of proteins or dimethyl sulfoxide. J Biomater Sci Polym Ed. 2013; 24(12):1484-97.
|28. Mamidi MK, Nathan KG, Singh G, Thrichelvam ST, Mohd Yusof NAN, Fakharuzi NA, et al. Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation. J Cell Biochem. 2012; 113(3):3153-64.
|29. Bahsoun S, Coopman K, Elizabeth C, Akam EC. The impact of cryopreservation on bone marrow‑derived mesenchymal stem cells:a systematic review. J Transl Med. 2019; 17:397. https://doi.org/10.1186/s12967-019-02136-7.
Tsymbaliuk V, Deryabina O, Shuvalova N, Verbovska S, Pichkur L, Oleksenko N, Kordium V. Cryopreservation of human Wharton’s jelly multipotent mesenchymal stromal cells with reduced concentration of dimethyl sulfoxide. Cell and Organ Transplantology. 2020; 8(1):51-57. doi:10.22494/cot.v8i1.109