Analysis of cryopreservation impact on mononuclear leukocyte metabolism

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Abstract

Studying the metabolic activity of mononuclear cells (MNCs) is crucial in biology and medicine. Cryopreservation is commonly used to store samples for long-term research, which helps minimize errors. However, the impact of low temperatures on MNCs metabolism remains understudied. The aim of this study was to investigate the effects of cryopreservation on glycolysis and oxidative phosphorylation in MNCs. Using the Seahorse XFe96 analyzer, we measured the metabolic parameters of cryopreserved MNCs via extracellular flux analysis. The results showed a significant decrease in the rate of oxidative phosphorylation in cryopreserved MNCs, without changes in MNC subset composition. Importantly, cryopreservation did not impact the rate of glycolysis. However, thawed cells exhibited reduced ability to increase metabolic rates in response to mitogenic stimulation. In conclusion, cryopreservation alters the metabolic profile of MNCs. To obtain reliable data on metabolic activity, the use of freshly isolated cells is preferable.

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About the authors

V. N. Ponomareva

Perm Federal Research Center, Ural Branch of the Russian Academy of Sciences; Perm State University

Author for correspondence.
Email: PonomarievaVN@yandex.ru

Institute of Ecology and Genetics of Microorganisms UB RAS, Department of Microbiology and Immunology

Russian Federation, 614081, Perm; 614068, Perm

V. V. Vlasova

Perm Federal Research Center, Ural Branch of the Russian Academy of Sciences

Email: PonomarievaVN@yandex.ru

Institute of Ecology and Genetics of Microorganisms UB RAS

Russian Federation, 614081, Perm

Е. V. Saidakova

Perm Federal Research Center, Ural Branch of the Russian Academy of Sciences; Perm State University

Email: PonomarievaVN@yandex.ru

Institute of Ecology and Genetics of Microorganisms UB RAS, Department of Microbiology and Immunology

Russian Federation, 614081, Perm; 614068, Perm

References

  1. Бабийчук Л. А., Михайлова О. А., Зубов П. М., Рязанцев В. В. 2013. Оценка стадий апоптоза и распределения фосфатидилсерина в мембране ядросодержащих клеток пуповинной и периферической крови при различных технологиях криоконсервации. Гены и клетки. Т. 8. № 4. С. 50. (Babijchuk L. A., Mykhailova O. O., Zubov P. M., Ryazantsev V. V. 2013. Evaluation of apoptosis stages and posphatidylserine distribution in membrane of cord and peripheral blood nucleated cells at various cryopreservation protocols. Genes and Cells. V. 8. P. 50.) https://doi.org/10.23868/gc121610
  2. Ващенко В. И., Чухловин А. Б., Петренко Г. И., Вильянинов В. Н., Багаутдинов Ш. М. 2015. Действие факторов замораживания на изменения внутриклеточного метаболизма при криоконсервации костного мозга человека. Вестник MAX. Т. 4. С. 91. (Vashchenko V. I., Сhuklovin A. B., Petrenko G. I., Vilyaninov V. N., Bagautdinov Ch. M. 2015. Impact of preserving agents upon on intracellular meтaboliс changes during cryoconservation of human bone marrow. J. IAR. V. 4. P. 91.)
  3. Кит О. И., Гненная Н. В., Филиппова С. Ю., Чембарова Т. В., Лысенко И. Б., Новикова И. А., Розенко Л. Я., Димитриади С. Н., Шалашная Е. В., Ишонина О. Г. 2023. Криоконсервация гемопоэтических стволовых клеток периферической крови в трансплантологии: современное состояние и перспективы. Кардиоваск. терапия и профилактика. Т. 22. № 11. С. 124. (Kit O. I., Gnennaya N. V., Filippova S. Yu., Chembarova T. V., Lysenko I. B., Novikova I. A., Rozenko L. Ya., Dimitriadi S. N., Shalashnaya E. V., Ishonina O. G. 2023. Cryostorage of peripheral blood hematopoietic stem cells in transplantology: current status and prospects. Cardiovascular Ther. Prevention. V. 22. P. 124.) https://doi.org/10.15829/1728-8800-2023-3691
  4. Нельсон Д., Кокс М. 2014. Основы биохимии Ленинджера. М.: БИНОМ. Лаборатория знаний. 636 с. (Nelson D. L., Cox M. M. 2014. Lehninger principles of biochemistry. M.: BINOM. Laboratory of knowledge. 636 p.)
  5. Савилова А. М., Чулкина М. М., Алексеев Л. П. 2013. Сравнительное исследование экспрессии мРНК интерлейкина-2 и рецептора интерлейкина-2α в лимфоцитах, активированных ФГА и КонА. Иммунология. Т. 34. № 2. С. 76. (Savilova A. M., Chulkina M. M., Alexeev L. P. 2013. Comparative investigation of interleukin-2 and interleukin-2 receptor alpha mRNA expression in lymphocytes activated by PHA or ConA. Immunol. V. 34. P. 76.)
  6. Anderson J., Toh Z. Q., Reitsma A., Do L. A.H., Nathanielsz J., Licciardi P. V. 2019. Effect of peripheral blood mononuclear cell cryopreservation on innate and adaptive immune responses. J. Immunol. Methods. V. 465. P. 61. https://doi.org/10.1016/j.jim.2018.11.006
  7. Chakraborty S., Khamaru P., Bhattacharyya A. 2022. Regulation of immune cell metabolism in health and disease: Special focus on T and B cell subsets. Cell Biol. Int. V. 46. P. 1729. https://doi.org/10.1002/cbin.11867
  8. Desdin-Mico G., Soto-Heredero G., Mittelbrunn M. 2018. Mitochondrial activity in T cells. Mitochondrion. V. 41. P. 51. https://doi.org/10.1016/j.mito.2017.10.006
  9. Fu Y., Dang W., He X., Xu F., Huang H. 2022. Biomolecular pathways of cryoinjuries in low-temperature storage for mammalian specimens. Bioengineering (Basel). V. 9. P. 545. https://doi.org/10.3390/bioengineering9100545
  10. Gaber T., Chen Y., Krauss P. L., Buttgereit F. 2019. Metabolism of T Lymphocytes in health and disease. Int. Rev. Cell Mol. Biol. V. 342. P. 95. https://doi.org/10.1016/bs.ircmb.2018.06.002
  11. Gualtieri R., Kalthur G., Barbato V., Di Nardo M., Adiga S. K., Talevi R. 2021. Mitochondrial dysfunction and oxidative stress caused by cryopreservation in reproductive cells. Antioxidants (Basel). V. 10. Art. ID: 337. https://doi.org/10.3390/antiox10030337
  12. Haider P., Hoberstorfer T., Salzmann M., Fischer M. B., Speidl W. S., Wojta J., Hohensinner P. J. 2022. Quantitative and functional assessment of the influence of routinely used cryopreservation media on mononuclear leukocytes for medical research. Int. J. Mol. Sci. V. 23. Art. ID: 1881. https://doi.org/10.3390/ijms23031881
  13. Len J. S., Koh W. S.D., Tan S. X. 2019. The roles of reactive oxygen species and antioxidants in cryopreservation. Biosci. Rep. V. 39. Art. ID: BSR20191601. https://doi.org/10.1042/BSR20191601
  14. Lomsadze G., Gogebashvili N., Enukidze M., Machavariani M., Intskirveli N., Sanikidze T. 2011. Alteration in viability and proliferation activity of mitogen stimulated Jurkat cells. Georgian Med. News. V. 9. P. 50.
  15. Makowski L., Chaib M., Rathmell J. C. 2020. Immunometabolism: from basic mechanisms to translation. Immunol. Rev. V. 295. P. 5. https://doi.org/10.1111/imr.12858
  16. Martikainen M. V., Roponen M. 2020. Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells. Toxicol. In Vitro. V. 67. Art. ID: 104918. https://doi.org/10.1016/j.tiv.2020.104918
  17. Mas-Bargues C., Garcia-Dominguez E., Borras C. 2022. Recent approaches to determine static and dynamic redox state-related parameters. Antioxidants (Basel). V. 11. Art. ID: 864. https://doi.org/10.3390/antiox11050864
  18. Patel C. H., Leone R. D., Horton M. R., Powell J. D. 2019. Targeting metabolism to regulate immune responses in autoimmunity and cancer. Nat. Rev. Drug Discov. V. 18. P. 669. https://doi.org/10.1038/s41573-019-0032-5
  19. Patil N. K., Bohannon J. K., Hernandez A., Patil T. K., Sherwood E. R. 2019. Regulation of leukocyte function by citric acid cycle intermediates. J. Leukoc. Biol. V. 106. P. 105. https://doi.org/10.1002/JLB.3MIR1118-415R
  20. Starkov A. A. 2008. The role of mitochondria in reactive oxygen species metabolism and signaling. Ann. N. Y. Acad. Sci. V. 1147. P. 37. https://doi.org/10.1196/annals.1427.015

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2. Fig. 1. Changes in basal oxygen consumption rates (OCR, a) and environment acidification rates (ECAR, b) by leukocytes before freezing and after cryopreservation. Data are presented as the area under the curve (AUC, conventional units). Means and standard errors of the means are shown. One-way analysis of variance was used to compare groups; multiple comparisons between groups were performed using the Tukey test. a: (**) — P < 0.01; (***) — P < 0.001; b — no significant differences were found.

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3. Fig. 2. Oxidative phosphorylation rates (OCR, a) and glycolysis rates (ECAR, b) in unstimulated (–) and phytohemagglutinin (PHA)-stimulated (+) mononuclear leukocytes. Data are presented as area under the curve (AUC). Means and standard errors of the means are shown. Student’s t-test was used to compare two groups. (*) — P < 0.05; (**) — P < 0.01.

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