Cell and Organ Transplantology. 2025; 13(1):14-21 (e2025131179).
DOI: 10.22494/cot.v13i1.179
L-carnitine as an antioxidant to improve the efficiency of cord blood hematopoietic progenitor cell cryopreservation
- Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine
Abstract
Cryopreservation of hematopoietic progenitor cells (HPCs) derived from cord blood is a critical step prior to transplantation, ensuring the long-term availability of viable cells for therapeutic use. However, oxidative stress during the freezing and thawing process can compromise cell survival and function, highlighting the need to improve cryopreservation protocols.
Purpose. This study aimed to evaluate the effectiveness of L-carnitine, an antioxidant, when added to DMSO-based cryoprotective solutions in enhancing the post-thaw survival and viability of human cord blood-derived HPCs under in vitro conditions simulating transfusion.
Materials and methods. Cord blood-derived nucleated cells, including CD45dimCD34+ HPCs, were collected after normal deliveries (n = 5). Cryopreservation was performed using DMSO at concentrations of 2.5 %, 5 %, and 7.5 %, supplemented with L-carnitine in the range of 1-50 mM. Cell viability was assessed immediately after thawing and following in vitro simulated transfusion (incubation in Hanks’ solution at 37 °C for 1 hour) using 7-aminoactinomycin D (7-AAD) staining and flow cytometry.
Results. The addition of L-carnitine to the cryopreservation medium significantly increased the viable yield of CD34⁺ cells, particularly at concentrations of 15-20 mM, both immediately after thawing and following incubation at physiological temperature. These results indicate that L-carnitine confers protective effects against oxidative stress-induced damage during cryopreservation.
Conclusion. The results obtained indicate that L-carnitine increases the efficiency of cryopreservation of human cord blood hematopoietic progenitor cells, contributing to an increase in their preservation and viability. The determined optimal cryoprotective medium may be useful for the development of new and improvement of existing protocols for the preservation of human cord blood-derived cellular components.
Key words: cord blood; hematopoietic progenitor cells; cell cryopreservation; L-carnitine
Full text PDF
| 1. Sun JM, Kurtzberg J. Cord blood for brain injury. Cytotherapy. 2015; 17(6):775-785. Available from: https://doi.org/10.1016/j.jcyt.2015.03.004 https://doi.org/10.1016/j.jcyt.2015.03.004 PMid:25800775 |
||||
| 2. Berglund S, Magalhaes I, Gaballa A, et al. Advances in umbilical cord blood cell therapy: the present and the future. Expert. Opin. Biol Ther. 2017; 17(6):691-699. Available from: https://doi.org/10.1080/14712598.2017.1316713 https://doi.org/10.1080/14712598.2017.1316713 PMid:28379044 |
||||
| 3. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood. 2013;122(4):491-498. Available from: https://doi.org/10.1182/blood-2013-02-453175 https://doi.org/10.1182/blood-2013-02-453175 PMid:23673863 PMCid:PMC3952633 |
||||
| 4. Chen G, Yue A, Ruan Z, Yin Y, Wang R, Ren Y, et al. Comparison of the Effects of Different Cryoprotectants on Stem Cells from Umbilical Cord Blood. Stem Cells Int. 2016; 5:1-7. Available from: https://doi.org/10.1155/2016/1396783 https://doi.org/10.1155/2016/1396783 PMid:26770201 PMCid:PMC4685149 |
||||
| 5. Verheijen M, Lienhard M, Schrooders Y, Clayton O, Nudischer R, Boerno S et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci. Rep. 2019; 9(1):4641. Available from: https://doi.org/10.1038/s41598-019-40660-0 https://doi.org/10.1038/s41598-019-40660-0 PMid:30874586 PMCid:PMC6420634 |
||||
| 6. Mommaerts K, Okawa S, Schmitt M, Kofanova O, Turner TR, Ben RN, et al. Ice recrystallization inhibitors enable efficient cryopreservation of induced pluripotent stem cells: A functional and transcriptomic analysis. Stem Cell Res. 2024; 81:103583. Available from: https://doi.org/10.1016/j.scr.2024.103583 https://doi.org/10.1016/j.scr.2024.103583 PMid:39467374 |
||||
| 7. Bennett D, Hanotaux J, Pasala AR, Hasan T, Hassan D, Shor R, et al. Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies. Cytotherapy. 2024; 6(5):482-489. Available from: https://doi.org/10.1016/j.jcyt.2024.02.006 https://doi.org/10.1016/j.jcyt.2024.02.006 PMid:38416086 |
||||
| 8. Kadekar D, Rangole S, Kale V, Limaye L. Conditioned medium from placental mesenchymal stem cells reduces oxidative stress during the cryopreservation of Ex Vivo expanded umbilical cord blood cells. PLoS One [Internet]. 2016; 11(10):e0165466. Available from: https://journals.plos.org/plosone/article?id = 10.1371/journal.pone.0165466 https://doi.org/10.1371/journal.pone.0165466 PMid:27780236 PMCid:PMC5079553 |
||||
| 9. Ray PD, Huang BW,Tsuji Y. Reactive Oxygen Species (ROS) Homeostasis and Redox Regulation in Cellular Signaling. Cellular Signalling. 2012; 24:981-990. Available from: https://doi.org/10.1016/j.cellsig.2012.01.008 https://doi.org/10.1016/j.cellsig.2012.01.008 PMid:22286106 PMCid:PMC3454471 |
||||
| 10. Xu X, Cowley S, Flaim C, James W, Seymour L, Cui Z. The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells. Biotechnology Progress. 2010; 26(3):827-837. Available from: https://doi.org/10.1002/btpr.368 https://doi.org/10.1002/btpr.368 PMid:20077485 PMCid:PMC3596802 |
||||
| 11. Fleury C, Mignotte B, Vayssière JL, Mitochondrial reactive oxygen species in cell death signaling. Biochimie, 2002; 84(2-3):131-141. Available from: https://doi.org/10.1016/S0300-9084(02)01369-X https://doi.org/10.1016/S0300-9084(02)01369-X PMid:12022944 |
||||
| 12. Makashova O, Babijchuk L, Zubov P, Zubova O. Optimization of the method of cryopreservation of nuclear cord blood cells with a unique combination of the cryoprotector DMSO and the antioxidant N-acetyl-L-cysteine. Probl Cryobiol Cryomed. 2016; 26(4):295-307. Available from: https://doi.org/10.15407/cryo26.04.295 https://doi.org/10.15407/cryo26.04.295 |
||||
| 13. Ye J, Li J, Yu Y. L-carnitine attenuates oxidant injury in HK-2 cells via ROS-mitochondria pathway. Regulatory Peptides. 2010; 161(1-3):58-66. Available from: https://doi.org/10.1016/j.regpep.2009.12.024 https://doi.org/10.1016/j.regpep.2009.12.024 PMid:20093144 |
||||
| 14. Surai PF. Antioxidant action of carnitine: molecular mechanisms and practical applications. EC Veterinary Science. 2015; 2(1):66-84. Available from: https://doi.org/10.13188/2325-4645.1000018 https://doi.org/10.13188/2325-4645.1000018 |
||||
| 15. Gülçin İ. Antioxidant and antiradical activities of l-carnitine. Life Sciences. 2006; 78(8):803-811. Available from: https://doi.org/10.1016/j.lfs.2005.05.103 https://doi.org/10.1016/j.lfs.2005.05.103 PMid:16253281 |
||||
| 16. Lohninger A, Pittner G, Pittner F. L-Carnitine: New Aspects of a Known Compound – A Brief Survey. Monatsh. Chem. 2005; 136(8):1255-1268. Available from: https://doi.org/10.1007/s00706-005-0339-2 https://doi.org/10.1007/s00706-005-0339-2 |
||||
| 17. Baust JM, Corwin W, Snyder KK, Van Buskirk R, Baust JG. Cryopreservation: Evolution of Molecular Based Strategies. In: Karimi-Busheri, F., Weinfeld, M. (eds) Biobanking and Cryopreservation of Stem Cells. Advances in Experimental Medicine and Biology. 2016; 951. Springer, Cham. Available from: https://doi.org/10.1007/978-3-319-45457-3_2 https://doi.org/10.1007/978-3-319-45457-3_2 PMid:27837551 |
||||
| 18. Babijchuk LO, Gryschenko VI. Gurina TM, et al. inventors; Institute for Problems of Cryobiology and Cryomedicine, assignee. [The method of cryopreservation of cord blood nucleated cells, including hematopoietic stem cells]. Patent of Ukraine 92227, 2010 October 11. Ukrainian. | ||||
| 19. Zubov P, Zubova O, Babijchuk L. Trolox Antioxidant as a Factor in Stabilization of Human Cord Blood Nucleated Cells During Cryopreservation. Probl Cryobiol Cryomed. 2023; 33(2):122-132. Available from: https://doi.org/10.15407/cryo33.02.122 https://doi.org/10.15407/cryo33.02.122 |
||||
| 20. Davis JM. Basic cell culture. A practical approach. Oxford: Oxford University Press; 2002. 382 p. | ||||
| 21. Zimmermann M, Meyer N. Annexin V/7-AAD staining in keratinocytes. Methods Mol. Biol. 2011; 740:57-63. Available from: https://doi.org/10.1007/978-1-61779-108-6_8 https://doi.org/10.1007/978-1-61779-108-6_8 PMid:21468968 |
||||
| 22. Maeshima Y, Makino H. Molecular mechanism of cell injury. Contrib. Nephrol. 2003; 139:32-43. Available from: https://doi.org/10.1159/000071735 https://doi.org/10.1159/000071735 PMid:12854317 |
||||
| 23. Heng BC, Ye CP, Liu H, Toh WS, Rufaihah AJ et al. Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis. J Biomed Sci 2006; 13:433-445. Available from: https://doi.org/10.1007/s11373-005-9051-9 https://doi.org/10.1007/s11373-005-9051-9 PMid:16374523 |
||||
| 24. Corwin WL, Baust JM, Baust JG, Van Buskirk RG. Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage. Cryobiology. 2014; 68(2): 215-226. Available from: https://doi.org/10.1016/j.cryobiol.2014.01.014 https://doi.org/10.1016/j.cryobiol.2014.01.014 PMid:24508650 PMCid:PMC4001798 |
||||
| 25. Bissoyi A., Nayak B, Pramanik K, Sarangi SK. Targeting Cryopreservation-Induced Cell Death: A Review. Biopreservation and biobanking. 2014; 12(1):23-34. Available from: https://doi.org/10.1089/bio.2013.0032 https://doi.org/10.1089/bio.2013.0032 PMid:24620767 |
||||
| 26. Scott KL, Lecak J, Acker JP. Biopreservation of red blood cells: past, present and future. Transfus Med Rev. 2005; 19(2):127-42. Available from: https://doi:10.1016/j.tmrv.2004.11.003. https://doi.org/10.1016/j.tmrv.2004.11.003 PMid:15852242 |
||||
| 27. Yu MH, Marquez-Curtis LA, Elliott JAW. Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers. Cryobiology. 2024; 115: 1-13. Available from: https://doi.org/10.1016/j.cryobiol.2024.104857 https://doi.org/10.1016/j.cryobiol.2024.104857 PMid:38350589 |
||||
| 28. Motta JPR, Gomes BE, Bouzas LF, Paraguassú-Braga FH, Porto LC. Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide. Cryobiology. 2010; 60(3): 301-7. Available from: https://doi.org/10.1016/j.cryobiol.2010.02.002 https://doi.org/10.1016/j.cryobiol.2010.02.002 PMid:20152822 |
||||
| 29. Reznick AZ, Kagan VE, Ramsay R, Tsuchiya M, Khwaja S, Serbinova EA, Packer L. Antiradical effects in L-propionyl carnitine protection of the heart against ischemia-reperfusion injury: The possible role of iron chelation. Archives of Biochemistry and Biophysics. 1992; 296(2):394-401. Available from: https://doi.org/10.1016/0003-9861(92)90589-o https://doi.org/10.1016/0003-9861(92)90589-O PMid:1321584 |
||||
| 30. Anderson EJ, Kypson AP, Rodriguez E, Anderson CA, Lehr EJ, Neufer PD. Substrate-specific derangements in mitochondrial metabolism and redox balance in the atrium of the type 2 diabetic human heart. J Am Coll Cardiol. 2009; 54(20):1891-8. Available from: https://doi.org/10.1016/j.jacc.2009.07.031 https://doi.org/10.1016/j.jacc.2009.07.031 PMid:19892241 PMCid:PMC2800130 |
||||
| 31. Manee-In S, Parmornsupornvichit S, Kraiprayoon S, Tharasanit T, Chanapiwat P, Kaeoket K. L-carnitine supplemented extender improves cryopreserved-thawed cat epididymal sperm motility. Asian-Australas J Anim Sci. 2014; 27(6): 791-6. Available from: https://doi:10.5713/ajas.2013.13565. https://doi.org/10.5713/ajas.2013.13565 PMid:25050016 PMCid:PMC4093175 |
||||
Zubova O, Zubov P. L-carnitine as an antioxidant to improve the efficiency of cord blood hematopoietic progenitor cell cryopreservation. Cell Organ Transpl. 2025; 13(1):14-21 (e2025131179). doi: https://doi.org/10.22494/cot.v13i1.179

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

