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Cell therapy is one of the new treatment methods in which mesenchymal stem/stromal cell (MSCs) transplantation is one of the cells widely used in this field. The results of MSCs application in the clinic prove their therapeutic efficacy. For this reason, many clinical trials have been designed based on the application of MSCs for various diseases, especially inflammatory disease and regenerative medicine. These cells perform their therapeutic functions through multiple mechanisms, including the differentiative potential, immunomodulatory properties, production of therapeutic exosomes, production of growth factors and cytokines, and anti-apoptotic effects. Exosomes are nanosized extracellular vesicles (EVs) that change target cell functions by transferring different cargos. The therapeutic ability of MSCs-derived exosomes has been demonstrated in many studies. However, some limitations, such as the low production of exosomes by cells and the need for large amounts of them and also their limited therapeutic ability, have encouraged researchers to find methods that increase exosomes' therapeutic potential. One of these methods is the spheroid culture of MSCs. Studies show that the three-dimensional culture (3DCC) of MSCs in the form of multicellular spheroids increases the therapeutic efficacy of these cells in laboratory and animal applications. In addition, the spheroid culture of MSCs leads to enhanced therapeutic properties of their exosomes and production rate. Due to the novelty of the field of using 3DCC MSCs-derived exosomes, examination of their properties and the results of their therapeutic application can increase our view of this field. This review discussed MSCs and their exosomes enhanced properties in spheroid culture.