Borozenets V. V

COMPARATIVE ASSESSMENT OF DIMETHYL SULFOXIDE TOXICITY IN 2D AND 3D CULTURES OF L929 CELL LINE

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About the author:

Borozenets V. V

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BIOLOGY

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Scientific article

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Monolayer and 3D cultures (spheroids, organoids) are widely used for in vitro drug testing, production of ther apeutic products, and investigation of disease mechanisms. Cryopreservation is essential for the long-term mainte nance of cell lines; Dimethyl sulfoxide (DMSO) is one of the most commonly used penetrating cryoprotectants for freezing and storing cells in cryobanks. At the same time, DMSO may exhibit cytotoxicity depending on its concentra tion, temperature, and incubation time. Cellular sensitivity to toxic agents differs between 2D and 3D models; how ever, direct comparisons of DMSO resistance between suspension cells and spheroids remain scarce. The purpose of this study was to perform a comparative analysis of DMSO cytotoxicity in 2D and 3D cultures of L929 cells. Following exposure to DMSO at concentrations of 5–15%, cell adhesion, proliferation, and the proportion of cells with mitotic abnormalities were evaluated. Model-specific sensitivity was identified: at 15% DMSO, the L929 monolayer culture exhibited sublethal effects, including reduced metabolic activity, decreased expression of adhesion molecules, and epigenetic alterations; whereas L929 cell spheroids were resistant to this concentration.

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1. Sharma K, Dey S, Karmakar R, Rengan AK. A comprehensive review of 3D cancer models for drug screening and translational research. Cancer Innov. 2023;3(1):e102. DOI: 10.1002/cai2.102.
2. Wei F, Wang S, Gou X. A review for cell-based screening methods in drug discovery. Biophys Rep. 2021;7(6):504-516. DOI: 10.52601/ bpr.2021. 210042.
3. Cardoso BD, Castanheira EMS, Lanceros-Méndez S, Cardoso VF. Recent Advances on Cell Culture Platforms for In Vitro Drug Screening and Cell Therapies: From Conventional to Microfluidic Strategies. Advanced healthcare materials. 2023;12(18):e2202936. DOI: 10.1002/ adhm.2022029 36.
4. Jandová M, Měřička P, Gregor J, Lánská M, Bezrouk A, Čížková D, et al. Post-thaw dimethyl sulfoxide reduction in autologous peripheral blood progenitor cell suspensions. Hematol Transfus Cell Ther. 2025;47(4):103965. DOI: 10.1016/j.htct.2025.103965.
5. Valeri CR, Pivacek LE. Effects of the temperature, the duration of frozen storage, and the freezing container on in vitro measurements in human peripheral blood mononuclear cells. Transfusion. 1996;36(4):303-308. DOI: 10.1046/j.1537-2995.1996.36496226141.x. 6.
6. spared by uncontrolled rate freezing. Bone marrow transplantation. 1999;23(5):497-503. DOI: 10.1038/sj.bmt.1701601.
7. Kaushal R, Jahan S, McGregor C, Pineault N. Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts. Cyto therapy. 2022;24(3):272-281. DOI: 10.1016/j.jcyt.2021.09.002.
8. Ehn K, Wikman A, Uhlin M, Sandgren P. Cryopreserved platelets in a non-toxic DMSO-free solution maintain hemostatic function in vitro. Int J Mol Sci. 2023;24(17):13097. DOI: 10.3390/ijms241713097.
9. Sangweni NF, Dludla PV, Chellan N, Mabasa L, Sharma JR, Johnson R. The implication of low dose dimethyl sulfoxide on mitochondrial function and oxidative damage in cultured cardiac and cancer cells. Molecules (Basel, Switzerland). 2021;26(23):7305. DOI: 10.3390/ molecules26237305.
10. Pakhomov O, Posokhov Y, Volkova N, Chernobai N, Bozhok G. Effects of cryoprotective agents on osmotic tolerance limit of testicular interstitial cells. Biophysical Bulletin. 2025;53:18-33. DOI: 10.26565/2075-3810-2025-53-02.
11. Giugliarelli A, Urbanelli L, Ricci M, Paolantoni M, Emiliani C, Saccardi R, et al. Evidence of DMSO-induced protein aggregation in cells. The journal of physical chemistry. 2016;120(27):5065-5070. DOI: 10.1021/acs.jpca.6b0 0178.
12. Ding Y, Liu S, Liu J, Jin S, Wang J. Cryopreservation with DMSO affects the DNA integrity, apoptosis, cell cycle and function of human bone mesenchymal stem cells. Cryobiology. 2024;114:104847. DOI: 10.1016/j.cryobiol.2024.104847.
13. Mitrus I, Smagur A, Fidyk W. 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 Trans plant. 2018;53:274-280. DOI: 10.1038/s41409-017-0056-6.
14. Bennett B, Hanotaux J, Pasala AR, Hasan T, Hassan D, Shor R, et al. Impact of lower concentrations of dimethyl sulfoxide on cryo preservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies. Cytotherapy. 2024;26(5):482-489. DOI: 10.1016/j.jcyt.2024.02.006.
15. Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells. 2021;13(9):1197-1214. DOI: 10.4252/wjsc.v13.i9.1197.
16. Ekpo MD, Boafo GF, Xie J, Liu X, Chen C, Tan S. Strategies in developing dimethyl sulfoxide (DMSO)-free cryopreservation protocols for biotherapeutics. Front Immunol. 2022;13:1030965. DOI: 10.3389/fimmu. 2022.1030965.
17. Awan M, Buriak I, Fleck R, Fuller B, Goltsev A, Kerby J, et al. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity?. Regenerative medicine. 2020;15(3):1463-1491. DOI: 10.2217/rme-2019-0145.
18. Goodman TT, Ng CP, Pun SH. 3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers. Biocon jug Chem. 2008;19(10):1951-9. DOI: 10.1021/bc800233a.
19. Dubois C, Dufour R, Daumar P, Aubel C, Szczepaniak C, Blavignac C, et al. Development and cytotoxic response of two proliferative MDA-MB-231 and non-proliferative SUM1315 three-dimensional cell culture models of triple-negative basal-like breast cancer cell lines. Oncotarget. 2017;8(56):95316-95331. DOI: 10.18632/oncotarget.20517.
20. Mikhail AS, Eetezadi S, Allen C. Multicellular tumor spheroids for evaluation of cytotoxicity and tumor growth inhibitory effects of nanomed icines in vitro: a comparison of docetaxel-loaded block copolymer micelles and Taxotere®. PloS one. 20138(4): e62630. DOI: 10.1371/ journal.pone.0062630.
21. Ehrhart F, Schulz JC, Katsen-Globa A, Shirley SG, Reuter D, Bach, et al. A comparative study of freezing single cells and spheroids: Towards a new model system for optimizing freezing protocols for cryobanking of human tumours, Cryobiology. 2009;58(2):119-127. DOI: 10.1016/j.cryobiol.2008. 11.005.
22. Moisieiev AI, Bozhok HA, Horina OL. Porivnialna kharakterystyka tryvymirnoho ta monosharovoho kultyvuvannia pereshchepliuvanoi linii fibroblastiv L929. Dopovidi Natsionalnoi akademii nauk Ukrainy. 2019;(8):93-101. DOI: 10.15407/dopovidi2019.08.093. [in Ukrainian]
23. Cannella V, Altomare R, Chiaramonte G, Di Bella S, Mira F, Russotto L, et al. Cytotoxicity evaluation of endodontic pins on L929 cell line. Biomed Res Int. 2019;2019:3469525. DOI: 10.1155/2019/3469525.
24. Sharma M, Sharma R, Jain DK. Nanotechnology based approaches for enhancing oral bioavailability of poorly water soluble antihyperten sive drugs. Scientifica. 2016;2016:8525679. DOI: 10.1155/2016/8525679.
25. Plaksina EM, Sidorenko OS, Bozhok GA. Cryopreservation of multicellular spheroids derived from newborn piglet adrenal glands. Prob lems of Cryobiology and Cryomedicine. 2017;27(4):322-333. DOI: 10.15407/cryo27. 04.322.
26. Tamarina IV, Bozhok GA, Gurina TM, Gubina NF, Bondarenko TP. Cryopreservation of newborn mice adrenal cell suspension. II. Effect caused by concentration of serum as component of cryoprotective medium. Problems of Cryobiology and Cryomedicine. 2013;22(1):66-74.
27. 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. DOI: 10.3329/bvet.v33i1.33307.
28. Meyer TP, Hofmann B, Zaisserer J, Jacobs VR, Fuchs B, Rapp S, et al. Analysis and cryopreservation of hematopoietic stem and progen itor cells from umbilical cord blood. Cytotherapy. 2006;8(3):265-276. DOI: 10.1080/14653240600735685.
29. Li X, Wang YK, Song ZQ, Du ZQ, Yang CX. Dimethyl sulfoxide perturbs cell cycle progression and spindle organization in porcine meiotic oocytes. PLoS One. 2016;11(6):e0158074. DOI: 10.1371/journal.pone.0158074.
30. Smalley KS, Lioni M, Herlyn M. Life isn’t flat: taking cancer biology to the next dimension. In vitro cellular & developmental biology. Animal. 2006;42(8-9):242-247. DOI: 10.1290/0604027.1.
31. Ncube KN, van den Bout I, Willers C, Gouws C, Cordier W. Utility of multicellular spheroids for investigating mechanisms of chemoresis tance in triple-negative breast cancer. Int. J. Mol. Sci. 2025;26:7503. DOI: 10.3390/ijms26157503.
32. Delarue M, Montel F, Vignjevic D, Prost J, Joanny JF, Cappello G. Compressive stress inhibits proliferation in tumor spheroids through a volume limitation. Biophysical journal. 2014;107(8):1821-1828. DOI: 10.1016/j. bpj.2014.08.031.
33. Pradhan S, Sperduto JL, Farino CJ, Slater JH. Engineered in vitro models of tumor dormancy and reactivation. J Biol Eng. 2018;12:37. DOI: 10.1186/s13036-018-0120-9.

Publication of the article:

«Bulletin of problems biology and medicine», 2026 Issue 1, 180, 162-171 pages, index UDC 576.32/.36.083.36:576.356

DOI:

10.29254/2077-4214-2026-1-180-162-171