Mesenchymal stem/stromal cells (MSC) are multipotent cells capable of influencing tissue regeneration, immunomodulation, and antiviral responses. During viral infection, they can reduce inflammation, promote tissue regeneration, and positively affect the course of disease through paracrine signaling and secretion of bioactive factors. Cytokine conditioning of MSCs mimics their natural microenvironment and can enhance their therapeutic efficacy. In this master’s thesis, we investigated whether MSC supernatants affect SARS-CoV-2 replication and assessed their potential toxicity to the VERO E6 cell line. The aim of this work was to evaluate the antiviral activity of the supernatants and to compare effects based on the mode of stimulation, tissue origin, and donor variability. Primary MSCs derived from different tissues and donors were incubated for 48 hours with interferon γ (IFN-γ) or with a combination of IFN-γ and tumor necrosis factor α (TNF-α). After stimulation, supernatants were collected, serially diluted, and first tested for cytotoxicity on VERO E6 cells using a colorimetric metabolic activity assay. Subsequently, supernatants were mixed with the virus and applied to cells to observe whether they inhibited viral replication. Results showed that supernatants from stimulated MSCs exerted minimal cytotoxic effects on VERO E6 cells; however, no supernatant induced a 50% reduction in cell viability. Cytotoxicity was most evident at the lowest dilutions and decreased with further dilution, while at the highest dilutions, cell viability was slightly higher compared to the control. Statistical analysis revealed differences in cytotoxicity depending on stimulation method, tissue origin, and donor, with the highest average cytotoxicity observed after stimulation with IFN-γ and TNF-α. Differences were more pronounced when donor and tissue related effects were considered. Using the selected assay, no inhibition of the cytopathic effect of the virus was detected at any dilution or in any sample. We demonstrated that MSCs from different tissue sources and donors produce distinct secretomes after cytokine stimulation. The applied qualitative method had limited sensitivity. For future studies, we propose more sensitive approaches to detect subtle antiviral effects, such as quantitative detection of the intracellular virus with monoclonal antibodies, shorter viral incubation times, concentration of supernatants, parallel testing of multiple SARS-CoV-2 variants, co-culture experiments of MSCs, immune cells and infected cells, and detailed analysis of secretome composition.
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