Figure legends
Figure 1. Preparation of m17.ASC spheroids. Macroscopic images of (A) micropillar arrays and (B) agarose-based microwell plate. Scale bars indicate 500 μm. (C) Top and (D) cross-sectional images of agarose-based microwell plate by microscopic observation. Scale bars indicate 300 μm. (E) A typical image of m17.ASC spheroid after 24-h incubation. Scale bar indicates 50 μm.
Figure 2. Size histogram of m17.ASC spheroids. m17.ASC spheroids were incubated for (A) 24 h, (B) 48 h, and (C) 72 h after seeding cells in an agarose-based microwell plate. The diameters of 100 spheroids were measured using a BZ-9000 microscope.
Figure 3. Characteristics of m17.ASC spheroids. (A) TGF-β secretion from m17.ASC spheroids. Monolayered m17.ASC cells (1.25 × 104 cells) or m17.ASC spheroids (11 spheroids: 1.25 × 104 cells) were seeded into a 12-well culture plate. After 24- and 48-h incubation, the amount of TGF-β in the supernatant was measured using an ELISA kit. (B) Arg1 and Ym1expression in peritoneal macrophages after co-culture with m17.ASC spheroids. Monolayered m17.ASC cells (1 × 105 cells) or m17.ASC spheroids (90 spheroids: 1 × 105 cells) were seeded into the upper wells of a 12-well Transwell plate, and 1 × 105 peritoneal macrophages were seeded into the lower wells. After 24-h incubation, mRNA (Arg1 and Ym1 ) expression in peritoneal macrophages was evaluated using real time RT-PCR. Results are normalized to mRNA levels of GAPDH . Data are expressed as the mean ± SD of three to four experiments.
Figure 4. Biodistribution of m17.ASC/Nluc spheroids after intravenous injection. (A) Relative light units of m17.ASC/Nluc spheroids in the plasma and tissues. m17.ASC/Nluc spheroids (135 spheroids: 1.5 × 105 cells/100 μL/mouse) or suspended m17.ASC/Nluc cells (1.5 × 105cells/100 μL/mouse) were intravenously injected in mice. Blood and tissues were collected at 24 h after intravenous injection, and relative light units of the plasma and supernatants of homogenized tissues were measured. (B) The numbers of m17.ASC/Nluc spheroids remaining in the lung. m17.ASC/Nluc spheroids (100 spheroids: 1.5 × 105cells/mouse) or suspended m17.ASC/Nluc cells (1.5 × 105 cells/mouse) were intravenously injected in mice. Lungs were collected at 6, 24, 48, 120, and 168 h after intravenous injection. The numbers of cells remaining in lungs were calculated by measuring the relative light units. (C) Typical images of CFSE-labeled m17.ASC spheroids in the lung. Suspended m17.ASC cells (1 × 105 cells) or m17.ASC spheroids (90 spheroids: 1 × 105 cells) were labeled with CFSE. Lungs were collected from euthanized mice 24 h after intravenous injection of cells. Cryosections of the lung were observed using a BZ-9000 microscope. Scale bar indicates 100 μm. Data are expressed as the mean ± SD of three to four experiments. *P < 0.05; statistically significant differences compared with suspension group.
Figure 5. Effect of m17.ASC spheroids on LPS-induced inflammatory mouse model. LPS was intraperitoneally administered to mice at 1 mg/kg. The mice were then bred on a hot plate (40 °C) for 1 h. m17.ASC spheroids (850 spheroids: 1 × 106 cells/100 μL/mouse) and suspended m17.ASC cells (1 × 106cells/100 μL/mouse) were intravenously injected. Blood was collected at 6 and 10 h after injection of cells. The (A) IL-6 and (B) TNF-α levels in plasma was measured using ELISA kits. Data are expressed as the mean ± SD of three to four experiments. *P < 0.05; statistically significant differences compared with the PBS group.