AbstractMacrophages are important cells of the innate immune system. They exhibit a high plasticity in phenotypes and play a major role in healing by initiating the early inflammatory reactions via the pro-inflammatory M1 phenotype. The anti-inflammatory M2 phenotype is assumed to induce regenerative processes and vascularization in subsequent tissue repair. Especially for regenerative processes, their interplay with multipotent human mesenchymal stromal cells (hMSCs) is decisive. Accordingly, in vitro co-culture models of these cell types are an important starting point for unraveling regenerative mechanisms. In our study, we compared direct co-culture, transwell-systems, and the use of conditioned medium to investigate the mitochondria transfer between the two cell types and the influence of hMSCs' presence on the phagocytic activity of macrophages. Using flow cytometry and fluorescence microscopy, we visualized the transfer of mitochondria in both directions: from hMSCs to macrophages and most notably also vice versa. Both cell types release mitochondria and internalize them in direct contact via tunneling nanotubes, as well as in indirect contact due to extracellular vesicles (EVs). Mitochondria were non-directionally released into the medium and could be transferred via conditioned medium. After three hours of direct and indirect co-culture, the majority of the cells showed a mitochondrial uptake. Co-cultivation also led to an increase of phagocytic activity of macrophages, with the highest phagocytic rate after 48 h and most pronounced in direct co-cultivation.