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Background  Mesenchymal stem cells (MSCs) have to be expanded in vitro to reach a sufficient cell dose for the treatment of various diseases. During the process of expansion, some obstacles remain to be overcome. The purpose of this study was to investigate the effects of storage solutions and heterogeneity on the behavior of MSCs in vitro and in vivo.    Methods  Umbilical cord MSCs (UC-MSCs) of similar sizes within normal ranges were suspended in three different storage solutions, phosphate buffer solution, normal saline, and Dulbecco’s modified Eagle medium. Then, the ultrastructure, viability, and safety of these cells were compared. Other two UC-MSC populations of different sizes were categorized based on their mean diameters. The ultrastructure, proliferation, immunosuppression, hepatic differentiation potential, and number of senescent cells were investigated and compared. The survival rates of mice after the infusion of UC-MSCs of different sizes were compared.    Results  For UC-MSCs suspended in different storage solutions, the cell apoptosis rates, ultrastructure, and survival rates of mice were similar, and no differences were observed. Cells with a diameter of 19.14 ± 4.89 μm were categorized as the larger UC-MSC population, and cells with a diameter of 15.58 ± 3.81 μm were categorized as the smaller population. The mean diameter of the larger UC-MSC population was significantly larger than that of the smaller UC-MSC population ( p  < 0.01). Smaller UC-MSCs had more powerful proliferation and immunosuppressive potential and a higher nucleus-cytoplasm ratio than those of large UC-MSCs. The number of cells positive for β-galactosidase staining was higher in the larger UC-MSC population than in the smaller UC-MSC population. The survival rates of mice receiving 1 × 10 6  or 2 × 10 6  smaller UC-MSCs were 100%, both of which were higher than those of mice receiving the same amounts of larger UC-MSCs ( p  < 0.01). The cause of mouse death was explored and it was found that some larger UC-MSCs accumulated in the pulmonary capillary in dead mice.    Conclusion  Different storage solutions showed no significant effects on cell behavior, whereas heterogeneity was quite prevalent in MSC populations and might limit cells application. Hence, it is necessary to establish a more precise standardization for culture-expanded MSCs.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13287-021-02450-2.

Cell heterogeneity, rather than the cell storage solution, affects the behavior of mesenchymal stem cells in vitro and in vivo