Medical Radiology and Radiation Safety. 2019. Vol. 64. No. 4. P. 18–24

DOI: 10.12737/article_5d11009f713799.54342353

A.A. Tsishnatti^1,2, M.V. Pustovalova¹, A.K. Grekhova¹, Yu.A. Bushmanov¹, T.A. Astrelina¹, I.V. Kobzeva¹, V.A. Nikitina¹, V.A. Brunchukov¹, D.Yu. Usupzhanova¹, I.M. Barabash¹, T.M. Blokhina¹, Yu.A. Fedotov¹, N.Yu. Vorobyova¹, A.S. Samoylov¹, A.N. Osipov¹

Influence of Ultra-High Dose Radiation on Cryopreserved Mesenchymal Stem Cells:
DNA Double-Strand Breaks and Proliferative Activity

1. A.I. Burnasyan Federal Medical Biophysical Center, Moscow, Russia. E-mail: Этот адрес электронной почты защищен от спам-ботов. У вас должен быть включен JavaScript для просмотра. ;
2. National Research Nuclear University MEPhI, Moscow, Russia

A.A. Tsishnatti – Technician;
M.V. Pustovalova – Researcher;
A.K. Grekhova – Junior Researcher;
Yu.A. Bushmanov – Head of Dep.;
T.A. Astrelina – Head of the Center for Biomedical Technologies, Dr. Sci. Med.;
I.V. Kobzeva – Head of the Center for Biomedical Technologies Cryobank, PhD Med.;
V.A. Nikitina – Leading Researcher, PhD Med., ISCT member;
V.A. Brunchukov – Junior Researcher;
D.Yu. Usupzhanova – Junior Researcher;
I.M. Barabash – Head of Dep.;
T.M. Blokhina – Researcher;
Yu.A. Fedotov – Researcher;
N.Yu. Vorobyova – Head of Lab., PhD Biol.;
A.S. Samoylov – Director General, Dr. Sci. Med., Prof. RAS;
A.N. Osipov – Head of Dep., Dr. Sci. Biol., Prof. RAS;

Abstract

Purpose: To conduct a comparative assessment of human mesenchymal stem cells (MSCs) exposed to ultrahigh doses of bremsstrahlung photon radiation at liquid nitrogen temperature (–196 °C) and room temperature (+22 °С) on the yield of residual DNA double-strand breaks (DSBs) and proliferative activity of thawed MSCs.

Material and methods: Isolation and cultivation of MSCs was carried out according to standard methods. Dimethyl sulfoxide (DMSO) at a final concentration of 10 % was used for cells cryopreservation. The cells were irradiated with bremsstrahlung photon radiation with photon nominal energy 5 MeV, using the UELR-10-100-T-100 accelerator (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of +22 °С and –196 °C. The immunocytochemical analysis of γH2AX foci (marker of DNA DSBs) was used for the assessment of the yield of residual DNA DSBs. The number of Ki67-positive cells (protein marker of cell proliferation) was analyzed for assessment of the cell proliferative activity.

Results: The results showed that48 hours after irradiation of MSCs at a dose of 50 Gy the number of residual γH2AX foci in the nuclei of MSCs irradiated at +22 °С was about 3.2 times (p = 0.0002) higher than in those irradiated at –196 °C. The analysis of the cell proliferative activity using Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of +22 °С completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, was significantly reduced, but some of the cells (3.5 ± 1.1 %) still retained the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lost their ability to proliferate, but partially retained the ability to adhere. The integral fluorescence of conjugated with the flurochrome γH2AX foci in MSCs irradiated at a dose of 500 Gy at a temperature of –196 °C was 1.8 times lower than that in MSCs irradiated at a temperature of +22 °С.

Conclusion: The results of the study indicate that MSCs cryopreserved in a medium containing 10 % DMSO irradiated at liquid nitrogen temperature (–196 °C) can tolerate the effects of exposure to high doses (up to 50 Gy) of ionizing radiation. However, there is a rather high yield of residual DNA DSBs and a very low proliferative activity, which makes cells unsuitable for use in clinical practice. It seems promising to use a quantitative analysis of γH2AX foci to assess genome damage and the functional state of cells irradiated in a cryopreserved state.

Key words: mesenchymal stem cells, cryopreservation, DNA double-strand breaks, cell proliferation, bremstrahlung, ultrahigh doses

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For citation: Tsishnatti AA, Pustovalova MV, Grekhova AK, Bushmanov YuA, Astrelina TA, Kobzeva IV, Nikitina VA, Brunchukov VA, Usupzhanova DYu, Barabash IM, Blokhina TM, Fedotov YuA, Vorobyova NYu, Samoylov AS, Osipov AN. Influence of Ultra-High Dose Radiation on Cryopreserved Mesenchymal Stem Cells: DNA Double-Strand Breaks and Proliferative Activity. Medical Radiology and Radiation Safety. 2019;64(4):18–24. (Russian).

DOI: 10.12737/article_5d11009f713799.54342353

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