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.