3. Results
3.1. Preparation of m17.ASC spheroids
Figure 1A and B show the appearance of the micropillar arrays and an agarose-based microwell plate, respectively. Figure 1C and D show microscopic images of the typical top and cross-sectional views of the agarose-based microwell plate. m17.ASC spheroids were successfully prepared using the agarose-based microwell plate 24 h after seeding the cells (Figure 1E). Figure 2 shows the size histogram of m17.ASC spheroids collected at 24, 48, and 72 h after incubation. The average diameter of the m17.ASC spheroids was approximately 170 μm, irrespective of the incubation period (Table 1). The number of m17.ASC cells ranged from 1,100 to 1,300 cells/spheroid and remained almost unchanged during the incubation period. The viability of the cells in spheroids was >90%. Based on these results, m17.ASC spheroids prepared from 24-h incubation were used in subsequent experiments.
3.2. Characteristics of m17.ASC spheroids
To evaluate the function of m17.ASC spheroids, the secretion of TGF-β, an anti-inflammatory cytokine, from m17.ASC spheroids or monolayered m17.ASC cells was measured. TGF-β secretion in both groups increased with time, and the amount of TGF-β secreted from m17.ASC spheroids tended to be higher than that from monolayered m17.ASC cells (Figure 3A). In addition, the effect of m17.ASC spheroids on the polarization of macrophages was evaluated, because MSCs are reported to modulate macrophages to the immunosuppressive M2-type and contribute to suppressing inflammation.[18] m17.ASC spheroids and monolayered m17.ASC cells tended to increase the expression ofArg1 and Ym1 , two M2-type macrophage markers, in peritoneal macrophages (Figure 3B). The expression of Arg1 in m17.ASC spheroids was higher than that in monolayered m17.ASC cells, although the difference was not statistically significant. These results indicate that spheroid formation enhanced the functions of m17.ASC cells, as previously reported in MSC spheroid formation.[19]
3.3. Tissue distribution of m17.ASC/Nluc spheroids and suspended m17.ASC/Nluc cells in mice
Figure 4A shows the tissue distribution of m17.ASC/Nluc spheroids and suspended m17.ASC/Nluc cells 24 h after intravenous injection in mice. m17.ASC/Nluc spheroids and suspended m17.ASC/Nluc cells showed high accumulation in the lung compared to other tissues. m17.ASC/Nluc spheroid accumulation in the lung was significantly higher than that in suspended m17.ASC/Nluc cells. Figure 4B shows the m17.ASC/Nluc cells remaining in the lung over time. At all the time points examined, the number of remaining cells was higher in the m17.ASC/Nluc spheroid group than that in the suspended m17.ASC/Nluc cell group. Furthermore, m17.ASC/Nluc spheroids were detected in the lung even at 168 h after injection, whereas suspended m17.ASC/Nluc cells almost completely disappeared after 48 h. Figure 4C shows typical images of the cryosections of the lung at 24 h after intravenous injection of CFSE-labeled m17.ASC spheroids or suspended CFSE-labeled m17.ASC cells. The fluorescence signal of CFSE-labeled m17.ASC spheroids was observed in most sections examined, whereas the fluorescence signal of suspended CFSE-labeled m.17ASC cells was hardly detected in any sections.
3.4. Effect of m17.ASC spheroids on LPS-induced inflammation in mice
Figure 5 shows the plasma concentrations of IL-6 and TNF-α in the LPS-induced inflammation mouse model. m17.ASC spheroids and suspended m17.ASC cells significantly reduced the LPS-induced increase in IL-6 and TNF-α levels. Importantly, m17.ASC spheroids were more effective than suspended m17.ASC cells in reducing TNF-α levels at 6 h. These results indicate that the therapeutic effect of m17.ASC spheroids was higher than that of suspended m17.ASC cells.