4. Discussion
Multicellular spheroids are useful cell formations in cell-based therapy because of the excellent cell–cell interactions, physical strength, and avoidance of anoikis, which is an apoptotic response to the absence of cell–matrix interactions. Although many reports have demonstrated improved cellular functions by spheroid formation in vitro ,[20-22] there have been few reports on the function or survival of multicellular spheroids after transplantation into animals. Our previous studies demonstrated that the survival of insulin-secreting cells was prolonged after transplantation into the subrenal capsule in mice[11] and that tumor colony formation in the lungs after intravenous injection in mice was accelerated by spheroid formation of tumor cells.[14] However, the function, survival, and tissue distribution of multicellular spheroids of MSCs after intravenous injection have been barely elucidated thus far. In particular, the quantitative comparison of suspended cells and spheroids after intravenous injection in terms of survival and tissue distribution has not been evaluated. Here, we showed that spheroid formation of MSCs improved their accumulation in the lungs and prolonged their survival after intravenous injection.
Several studies have reported that spheroid formation significantly increases cytokine release from MSCs.[10,23] Here, TGF-β release from m17.ASC spheroids was slightly higher than that from monolayered m17.ASC cells (Figure 3A). Additionally, the release of other cytokines from m17.ASC spheroids may have been improved, because the expression of Arg1 , an M2-type marker, by peritoneal macrophages was greatly increased by co-culture with m17.ASC spheroids (Figure 3B). As the MSC-induced polarization of macrophages to the M2-type has been reported to be related to an anti-inflammatory function,[24] the results of the present study strongly suggest the therapeutic potential of MSC spheroids for inflammatory diseases. In addition to cytokine expression, spheroid formation of MSCs can be an effective means of cell-based therapy.
The size of multicellular spheroids is an important parameter in determining the tissue distribution of cells after intravenous injection. This is because the diameter of microvessels in the lung, the first-pass organ after intravenous injection, is approximately 10 μm, and most cells transiently form the embolus in the lung after intravenous injection.[25] Our previous study showed that intravenously injected multicellular spheroids of adenocarcinoma cells with a diameter of approximately 170 μm effectively formed tumor colonies in the lung compared to suspended cells.[14] Therefore, m17.ASC spheroids of approximately 170 μm diameter were prepared in this study (Table 1), and intravenously injected m17.ASC spheroids efficiently accumulated and remained in the lung.
LPS-induced inflammatory mouse models are frequently used as systemic inflammatory disease models through endotoxin shock, which causes an increase in the release of inflammatory cytokines such as IL-6 in the plasma and multiple organ failures.[26] Many reports have demonstrated that MSC administration effectively suppresses inflammation and shows a therapeutic effect in LPS-induced inflammatory mouse models.[27,28] Compared to m17.ASC cells, m17.ASC spheroids more efficiently suppressed the plasma levels of inflammatory cytokines in LPS-induced inflammation mouse models (Figure 5). This result reflects the prolonged survival of the m17.ASC spheroids after intravenous injection (Figure 4).
In conclusion, we demonstrated that spheroid formation of m17.ASC cells improved the delivery efficiency of the cells to the lung and prolonged their survival after intravenous injection. Additionally, compared to suspended cells, the m17.ASC spheroids showed excellent therapeutic effect in the inflammatory mouse models.