L-carnitine as an antioxidant to improve the efficiency of cord blood hematopoietic progenitor cell cryopreservation

Home/2025, Vol. 13, No. 1/L-carnitine as an antioxidant to improve the efficiency of cord blood hematopoietic progenitor cell cryopreservation

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

Zubova O., Zubov P.

  • 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

 

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