Trehalose: effect on cryopreservation of umbilical cord blood-derived hematopoietic stem cells
Abstract
Introduction: Concerns over dimethyl sulfoxide (DMSO) toxicity, related to adverse reactions after hematopoietic stem cells (HSCs) therapy, warrant the development of an optimized DMSO-free or DMSO-reduced cryopreservation protocol for the quality and safety of HSCs.
In this regard, the ideal concentration of trehalose, as a non-toxic natural cryoprotectant, is still an area of research. Based on the outcome of our previous study on lower concentrations of trehalose, the present study was focused on evaluating its cryoprotective efficacy at an increased concentration (0.5 M) on HSCs compared to 10% DMSO. This is a laboratory-based experimental study.
Material and methods: The separated mononuclear cells collected from umbilical cord blood were set for culture up to two passages to get HSCs. The two different concentrations of trehalose, with and without 5% DMSO, were considered as freezing media for the preservation of the harvested HSCs in a slow freezing set up. Two sequential functional assays, viability followed by hematopoietic colony-forming unit assay, were performed with post-thawed cells of freezing media used in this study. Seventeen cord blood samples were selected.
Results: Study results revealed 0.5 M trehalose and DMSO 5% showed the highest viability of 91.8 ±2.8% of HSCs. 5% DMSO inclusion to trehalose (0.5 M) ameliorated hematopoietic colonies such as erythroid and myeloid colonies with no significant difference from that of 10% DMSO.
Conclusion: 0.5 M trehalose has proved to be a better concentration than 10% DMSO alone. This experimental study needs further transplantation-based clinical trials using post-thawed cells to ensure the safety of preserved HSCs from cord blood and other sources.
Keywords: DMSOcord bloodtrehalosecryoprotective media
References
- Hornberger K, Yu G, McKenna D, et al. Cryopreservation of hematopoietic stem cells: emerging assays, cryoprotectant agents, and technology to improve outcomes. Transfus Med Hemother. 2019; 46(3): 188–196.
- Ruiz-Delgado GJ, Mancías-Guerra C, Tamez-Gómez EL, et al. Dimethyl sulfoxide-induced toxicity in cord blood stem cell transplantation: report of three cases and review of the literature. Acta Haematol. 2009; 122(1): 1–5.
- Shu Z, Heimfeld S, Gao D. Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplantation. 2013; 49(4): 469–476.
- Hirata Y, Kishino K, Onozaki F, et al. Use of cryoprotectant-depleted allogeneic peripheral blood stem cells for transplantation. Hematology. 2011; 16(4): 221–224.
- Scerpa MC, Daniele N, Landi F, et al. Automated washing of human progenitor cells: evaluation of apoptosis and cell necrosis. Transfus Med. 2011; 21(6): 402–407.
- Abonnenc M, Pesse B, Tissot JD, et al. Automatic washing of thawed haematopoietic progenitor cell grafts: a preclinical evaluation. Vox Sang. 2017; 112(4): 367–378.
- Aerts-Kaya F, Koca G, Sharafi P, et al. Automated washing of long-term cryopreserved peripheral blood stem cells promotes cell viability and preserves CD34+ cell numbers. Bone Marrow Transplant. 2018; 53(9): 1225–1227.
- Galmes A, Gutiérrez A, Sampol A, et al. Long-term hematological reconstitution and clinical evaluation of autologous peripheral blood stem cell transplantation after cryopreservation of cells with 5% and 10% dimethylsulfoxide at -80 degrees C in a mechanical freezer. Haematologica. 2007; 92(7): 986–989.
- Rowley SD, Feng Z, Chen L, et al. A randomized phase III clinical trial of autologous blood stem cell transplantation comparing cryopreservation using dimethylsulfoxide vs dimethylsulfoxide with hydroxyethylstarch. Bone Marrow Transplant. 2003; 31(11): 1043–1051.
- Hayakawa J, Joyal EG, Gildner JF, et al. 5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO. Transfusion. 2010; 50(10): 2158–2166.
- 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.
- Stolzing A, Naaldijk Y, Fedorova V, et al. Hydroxyethylstarch in cryopreservation — mechanisms, benefits and problems. Transfusion and Apheresis Science. 2012; 46(2): 137–147.
- https://www.sciencedirect.com/topics/medicine-and-dentistry/hydroxyethyl-starch (March 26, 2020).
- https://www.sciencedirect.com/topics/neuroscience/dextran-40 (March 26, 2020).
- Jain NK, Roy I. Effect of trehalose on protein structure. Protein Sci. 2009; 18(1): 24–36.
- Martinetti D, Colarossi C, Buccheri S, et al. Effect of trehalose on cryopreservation of pure peripheral blood stem cells. Biomed Rep. 2017; 6(3): 314–318.
- Mantri S, Kanungo S, Mohapatra PC. Cryoprotective effect of disaccharides on cord blood stem cells with minimal use of DMSO. Indian J Hematol Blood Transfus. 2015; 31(2): 206–212.
- Page KM, Zhang L, Mendizabal A, et al. Total colony-forming units are a strong, independent predictor of neutrophil and platelet engraftment after unrelated umbilical cord blood transplantation: a single-center analysis of 435 cord blood transplants. Biol Blood Marrow Transplant. 2011; 17(9): 1362–1374.
- Keong CK, Nadarajah VD, Lee TJu. Development of a purification method of pure primary lymphocytes for cell viability assays. Malays J Med Sci. 2007; 14(1): 38–45.
- Mahmut N, Katayama Y, Takenaka K, et al. Analysis of circulating hematopoietic progenitor cells after peripheral blood stem cell transplantation. Int J Hematol. 1999; 69(1): 36–42.
- Luzardo MC, Amalfa F, Nuñez AM, et al. Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers. Biophys J. 2000; 78(5): 2452–2458.
- Mfarrej B, Bouchet G, Couquiaud J, et al. Pre-clinical assessment of the Lovo device for dimethyl sulfoxide removal and cell concentration in thawed hematopoietic progenitor cell grafts. Cytotherapy. 2017; 19(12): 1501–1508.
- Aerts-Kaya F, Koca G, Sharafi P, et al. Automated washing of long-term cryopreserved peripheral blood stem cells promotes cell viability and preserves CD34+ cell numbers. Bone Marrow Transplant. 2018; 53(9): 1225–1227.
- Mitrus I, Smagur A, Fidyk W, et al. Reduction of DMSO concentration in cryopreservation mixture from 10% to 7.5% and 5% has no impact on engraftment after autologous peripheral blood stem cell transplantation: results of a prospective, randomized study. Bone Marrow Transplant. 2018; 53(3): 274–280.
- Scheinkönig C, Kappicht S, Kolb HJ, et al. Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells. Bone Marrow Transplant. 2004; 34(6): 531–536.
- Morgenstern DA, Ahsan G, Brocklesby M, et al. Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes. Br J Haematol. 2016; 174(6): 942–951.
- Chevaleyre J, Rodriguez L, Duchez P, et al. A novel procedure to improve functional preservation of hematopoietic stem and progenitor cells in cord blood stored at +4°c before cryopreservation. Stem Cells Dev. 2014; 23(15): 1820–1830.
