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

Abstract

Purpose: To conduct a comparative assessment of the effect of irradiation of human mesenchymal stem cells (MSC)at ultrahigh doses at liquid nitrogen temperature (–196 °C) and room temperature (+22 °C) on the yield of “residual” DNA double-strand breaks (DSB) and proliferative activity of thawed MSC. Material and methods: Isolation and cultivation of MSC 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, the accelerator UELR-10-100-T-100 (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of 22 °C and –196 °C. The yield of “residual” DNA DSB was assessed using an immunocytochemical analysis of the foci of the protein-marker DSB – γH2AX. To assess the proliferative activity, the number of Ki67 (protein marker of cell proliferation) of positive cells was analyzed. Results: The results of γH2AX foci assessment in MSCs after 48 hours irradiation at a dose of 50 Gy showed that the number of “residual” γH2AX foci in the MSC nuclei irradiated at 22 °C is about 3.2 times (p = 0.0002) higher than in the MSC nuclei irradiated at –196 °C. Analysis of the proliferative activity of cells using the molecular marker of cell proliferation of the Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of 22 °C, completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, is significantly reduced, but some of the cells (3.5 ± 1.1 %) still retain the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lose their ability to proliferate, but partially retain the ability to adhere. The integral fluorescence conjugated with the flurochrome of antibodies to γH2AX of MSC nuclei irradiated at a dose of 500 Gy at a temperature of –196 °C is 1.8 times lower than that of nuclei irradiated at a temperature of 22 °C. Conclusion: The results of the studies indicate that cryopreserved MSCs irradiated at liquid nitrogen temperature (–196 °C) in a preservation medium containing 10 % DMSO can tolerate the effects of ionizing radiation in large doses (up to 50 Gy). However, there is a rather high yield of “residual” DSB DNA and a very low proliferative activity, which makes them unsuitable 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.