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

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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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