Integrated Low-DMSO Cryopreservation and Expansion Strategies Enhance Cord Blood CD34⁺ Stem Cell Recovery and Function
DOI:
https://doi.org/10.65329/wjeb.v14.01.04Keywords:
Cord blood bank; CD34+, cryopreservation, hematopoietic stem cells, plant hormones.Abstract
Umbilical cord blood (UCB) is a source of CD34+ hematopoietic stem cells (HSCs). The main challenges in handling them are the cytotoxicity of 10% dimethyl sulfoxide (DMSO) cryoprotection and insufficient cell number from a single cord blood unit. To address them, UCB-derived mononuclear cells (MNCs) were isolated by Ficoll density gradient centrifugation and cryopreserved in different formulations: 2.5% or 5% DMSO, each alone or supplemented with trehalose (25 µg/mL) or ascorbic acid (80 µg/mL). Post-thaw, viable MNC counts were assessed using trypan blue viability, and CD34+ immunocytochemistry was performed. To overcome the second challenge, MNCs were cultured for 7 days with 5%, 10%, or 25% placental extract, or for 4 days with 4 plant hormones, including kinetin, indole-3-acetic acid, naphthaleneacetic acid, and gibberellic acid at 2.5 and 5 mg/L. The MTT assay was used to evaluate the cytotoxicity. The results showed that 5% DMSO with trehalose yielded the highest post-thaw MNC count (4.70 ± 0.10 × 106 cells/mL) and viability of 54.50 ± 0.28%, whereas 5% DMSO with ascorbic acid produced the highest CD34+ retention (81.16 ± 0.60%), both significantly higher than in 2.5% DMSO formulations (P < 0.05). 5% placental extract increased cloning efficiency 5.7-fold compared with the control (P < 0.05). The hormones used depleted differentiated MNCs while preserving CD34+ content at 89-96%. These findings establish 5% DMSO with trehalose or ascorbic acid as an alternative to conventional 10% DMSO protocols, and 5% placental extract as a cost-effective HSC expansion supplement with direct translational importance for cord blood banking and transplantation medicine.
References
[1] Mayani H, Lansdorp PM. (1998) Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells 16(3): 153–165. doi: https://doi.org/10.1002/stem.160153 , PMID: 9617891.
[2] Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, et al. (1998) Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 339(22):1565–1577. doi: https://doi.org/10.1056/NEJM199811263392201, PMID: 9828244
[3] Mayani H. (2024) Umbilical Cord Blood Hematopoietic Cells: From Biology to Hematopoietic Transplants and Cellular Therapies. Arch Med Res 55(6): 103042. doi: https://doi.org/10.1016/j.arcmed.2024.103042 , PMID: 39003965.
[4] Zenhäusern R, Tobler A, Leoncini L, Hess OM, Ferrari P. (2000) Fatal cardiac arrhythmia after infusion of dimethyl sulfoxide-cryopreserved hematopoietic stem cells in a patient with severe primary cardiac amyloidosis and end-stage renal failure. Ann Hematol 79(9): 523–526. doi: https://doi.org/10.1007/s002770000186, PMID: 11043425 .
[5] Zambelli A, Poggi G, Da Prada G, Pedrazzoli P, Cuomo A, et al. (1998). Clinical toxicity of cryopreserved circulating progenitor cells infusion. Anticancer Res 18(6B):4705–4708. PMID: 9891544.
[6] Taherian MM, Abdoos P, Taherian MH, Ghorbanian F, Saltanatpour Z, Alizadeh A. (2024) Cryopreservation of Stem Cells in Tissue Engineering and Regenerative Medicine. J Appl Biotechnol Rep 11(3):1359-1370. doi: https://doi.org/10.30491/jabr.2022.331200.1501
[7] Sun WQ, Davidson P. (1998) Protein inactivation in amorphous sucrose and trehalose matrices: effects of phase separation and crystallization. Biochim Biophys Acta 1425(1):235–244. https://doi.org/10.1016/s0304-4165(98)00076-2 , PMID: 9813347
[8] Sabbah MA, Shamkhi NF, Alwachi SN. (2011) Trehalose and ascorbic acid improves the Cryopreservation of umbilical Cord Blood hematopoietic stem Cells (CD34+) with low Concentrations of Dimethylsulfoxide. Iraqi J Cancer Med Genet 4(1):57-62. https://doi.org/10.29409/ijcmg.v4i1.58
[9] Motta JP, Paraguassú-Braga FH, Bouzas LF, Porto LC. (2014) Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood. Cryobiology 68(3): 343–348. https://doi.org/10.1016/j.cryobiol.2014.04.007 , PMID: 24769312.
[10] Mantri S, Satpathy AK, Mohapatra PC. (2021) Trehalose: effect on cryopreservation of umbilical cord blood-derived hematopoietic stem cells. Acta Haematol Pol 52(2):121–126. https://doi.org/10.5603/AHP.2021.0018.
[11] Buettner GR. (1993) The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch Biochem Biophys 300(2):535–543. https://doi.org/10.1006/abbi.1993.1074 ,PMID: 8434935.
[12] Limaye LS, Kale VP. (2001) Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium. J Hematother Stem Cell Res 10(5):709–718. https://doi.org/10.1089/152581601753193931, PMID: 11672518
[13] Mantri S, Kanungo S, Mohapatra PC. (2015) Cryoprotective Effect of Disaccharides on Cord Blood Stem Cells with Minimal Use of DMSO. Indian J Hematol Blood Transfus 31(2):206–212. https://doi.org/10.1007/s12288-014-0352-x, PMID: 25825559
[14] Liu B, Tao C, Wu Z, Yao H, Wang DA. (2022) Engineering strategies to achieve efficient in vitro expansion of haematopoietic stem cells: development and improvement. J Mater Chem B 10(11): 1734–1753. https://doi.org/10.1039/d1tb02706a , PMID: 35191442
[15] Kadekar D, Kale V, Limaye L. (2015) Differential ability of MSCs isolated from placenta and cord as feeders for supporting ex vivo expansion of umbilical cord blood derived CD34(+) cells. Stem Cell Res Ther 6: 201. https://doi.org/10.1186/s13287-015-0194-y, PMCID: PMC4617445
[16] Teleb RS, Abdul-Hafez A, Othman A, Ahmed AE, Elsaid AA, et al. (2023) Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness. Cells 12(2), 250. https://doi.org/10.3390/cells12020250, PMCID: PMC9857343
[17] Wang L, Cheng J, Lin F, Liu S, Pan H, et al. (2019) Ortho-Topolin Riboside Induced Differentiation through Inhibition of STAT3 Signaling in Acute Myeloid Leukemia HL-60 Cells. Turk J Haematol 36(3):162–168. https://doi.org/10.4274/tjh.galenos.2019.2019.0020, PMCID: PMC6682775
[18] Park E, Ciofani M (2025) Th17 cell pathogenicity in autoimmune disease. Exp Mol Med 57(9):1913–1927. https://doi.org/10.1038/s12276-025-01535-9, PMCID: PMC12508148
[19] Surbek DV, Schönfeld B, Tichelli A, Gratwohl A, Holzgreve W. (1998) Optimizing cord blood mononuclear cell yield: a randomized comparison of collection before vs after placenta delivery. Bone Marrow Transplant 22(3):311–312. https://doi.org/10.1038/sj.bmt.1701315, PMID: 9720751.
[20] Solves P, Moraga R, Saucedo E, Perales A, Soler MA, et al. (2003) Comparison between two strategies for umbilical cord blood collection. Bone Marrow Transplant 31(4):269–273. https://doi.org/10.1038/sj.bmt.1703809, PMID: 12621461.
[21] Sutherland DR, Keating A. (1992) The CD34 antigen: structure, biology, and potential clinical applications. J Hematother 1(2):115–129. https://doi.org/10.1089/scd.1.1992.1.115, PMID: 1285404
[22] Scott KL, William N, Acker JP. (2022) The response of a human haematopoietic cell line to trehalose-loaded liposomes and their effect on post-thaw membrane integrity. Cryobiology 106:160–163. https://doi.org/10.1016/j.cryobiol.2022.03.005, PMID: 35413361
[23] Jafar H, Abuarqoub D, Ababneh N, Hasan M, Al-Sotari S, et al. (2019) hPL promotes osteogenic differentiation of stem cells in 3D scaffolds. PloS one 14(5): e0215667. https://doi.org/10.1371/journal.pone.0215667, PMCID: PMC6504042
[24] Sakurai M, Ishitsuka K, Becker HJ, Yamazaki S. (2024) Ex vivo expansion of human hematopoietic stem cells and clinical applications. Cancer Sci 115(3):698–705. https://doi.org/10.1111/cas.16066, PMCID: PMC10921004.
[25] Meaker GA, Wilkinson AC. (2024) Ex vivo hematopoietic stem cell expansion technologies: recent progress, applications, and open questions. Exp Hematol 130:104136. https://doi.org/10.1016/j.exphem.2023.12.001, PMCID: PMC11511678
[26] Cilloni D, Garau D, Regazzi E, Sammarelli G, Savoldo B, et al. (1999) Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing. Bone Marrow Transplant 23(5):497–503. https://doi.org/10.1038/sj.bmt.1701601, PMID: 10100565.
[27] Yang H, Acker JP, Cabuhat M, Letcher B, Larratt L, McGann LE. (2005) Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment. Bone marrow transplant 35(9):881–887. https://doi.org/10.1038/sj.bmt.1704926, PMID: 15778729.
[28] Maas RGC, Lee S, Harakalova M, Snijders Blok CJB, Goodyer WR, et al. (2021) Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes. STAR Protoc 2(1):100334. https://doi.org/10.1016/j.xpro.2021.100334, PMCID: PMC7881265
[29] Zhang XB, Li K, Yau KH, Tsang KS, Fok TF, et al. (2003) Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic-SCID repopulating cells. Transfusion 43(2):265–272. https://doi.org/10.1046/j.1537-2995.2003.00301.x, PMID: 12559024.
[30] Mondal B. (2009). A simple method for cryopreservation of MDBK cells using trehalose and storage at -80 degrees C. Cell Tissue Bank 10(4): 341–344. https://doi.org/10.1007/s10561-009-9130-7, PMID: 19381873.
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