2. Materials and methods
2.1. Materials
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo, Japan). Trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.25% trypsin and 1 mM EDTA) and trypan blue were purchased from Nacalai Tesque (Kyoto, Japan). SYBR Green PCR Master Mix was obtained from Gibco Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Agarose S was purchased from Nippon Gene (Toyama, Japan). Isoflurane was purchased from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). Hygromycin B Gold was purchased from InvivoGen (San Diego, CA, USA). Hank’s Balanced Salt solution (HBSS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of the highest grade commercially available.
2.2. Animals
Male FVB/N mice (6–8 weeks old) were purchased from CLEA Japan Inc. (Tokyo, Japan). All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for the animal experiments were approved by the Animal Experimentation Committee of the Tokyo University of Science.
2.3. Cell culture
The murine adipose-derived MSC line m17.ASC was purchased from DS Pharma Biomedical (Osaka, Japan) and grown in DMEM supplemented with 20% inactivated FBS and penicillin-streptomycin-glutamine mixed solution. Peritoneal macrophages were collected from male FVB/N mice. In detail, cold phosphate-buffered saline (PBS, 5 mL) was injected into the abdominal cavity of euthanized FVB/N mice using a 26G needle and syringe, and the PBS containing peritoneal cells was collected after rubbing the abdomen. The collected PBS was centrifuged at 300 × gfor 3 min to sediment and obtain the peritoneal cells. The cells were grown in DMEM supplemented with 10% inactivated FBS and penicillin-streptomycin-glutamine mixed solution. After 24-h incubation, adherent cells were used as peritoneal macrophages. All cells were maintained in a humidified atmosphere containing 5% CO2at 37 °C.
2.4. Preparation of agarose-based microwell plates
Micropillar arrays fabricated using a 3D printing system (Objet Geometries, Rehovot, Israel)[15] were used as molds for agarose-based microwell plates. Agarose was dissolved in distilled water at a final concentration of 3% (w/v), heated in a microwave oven, and poured into the micropillar arrays. After gelation of the agarose solution by cooling for 30 min at approximately 20 °C, the micropillar arrays were removed from the agarose gel. The agarose-based microwell plate was cut with a cutter to adjust the size to fit a well in a six-well cell culture plate. Microwell plates in culture plates were sterilized by ultraviolet irradiation prior to cell seeding. The structures of the micropillar arrays and agarose-based microwell plates were imaged using a digital camera (IXY 200F, Canon, Tokyo, Japan) and BZ-9000 microscope (Keyence, Osaka, Japan).
2.5. Preparation of m17.ASC spheroids
m17.ASC cells (2 × 106 cells) suspended in a cell culture medium were added to an agarose-based microwell plate in a six-well plate, and the culture plates were shaken for 2 h. The medium was then replaced with fresh medium, and the cells were incubated at 37 °C with rotary shaking at 60 rpm. At 24, 48, and 72 h of incubation, spheroids formed in microwells were collected using a micropipette.
2.6. Evaluation of m17.ASC spheroids
The diameter of the m17.ASC spheroids was measured by observation under a BZ-9000 microscope (Keyence) using the BZ-9000 analyzer software. The average diameter was calculated as the average diameter of 100 spheroids. The number and viability of cells in spheroids were evaluated by trypan blue staining after dispersing 20 spheroids in trypsin-EDTA solution.
2.7. Transforming growth factor (TGF)-β release from m17.ASC spheroids
To evaluate cytokine release from m17.ASC spheroids, monolayered m17.ASC cells (1.25 × 104 cells) or m17.ASC spheroids (11 spheroids: approximately 1.25 × 104 cells, 24-h incubation) were seeded into a 12-well culture plate. After 24 and 48 h of incubation, the supernatant was collected and centrifuged at 2,500 rpm for 5 min to remove cell debris. The concentration of TGF-β in the supernatant was measured using a TGF beta-1 Human/Mouse Enzyme-Linked Immunosorbent Assay kit (Thermo Fisher Scientific).
2.8. Polarization of macrophages by co-culture with m17.ASC spheroids
To evaluate the polarization of macrophages by co-culture with m17.ASC spheroids, monolayered m17.ASC cells (1 × 105 cells) or m17.ASC spheroids (90 spheroids: approximately 1 × 105 cells) were added to the upper wells of a 12-well Transwell plate (Corning, NY, USA), and 1 × 105peritoneal macrophages were seeded into the lower wells. After 24-h incubation, total RNA was extracted from peritoneal macrophages using Sepasol RNA I Super G (Nacalai Tesque). mRNA was reverse-transcribed to cDNA using qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). To quantify the gene of interest, cDNA samples were amplified using Thunderbird SYBR qPCR Mix (Toyobo) and specific primers with the CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences used were as follows: GAPDH (F) 5’-AGGTCGGTGTGAACGGATTTG-3’, GAPDH (R) 5’-TGTAGACCATGTAGTTGAGGTCA-3’; Arg1 (F) 5’-TGGCTTGCGAGACGTAGAC-3’, Arg1 (R) 5’-GCTCAGGTGAATCGGCCTTTT-3’;Ym1 (F) 5’-TTATCCTGAGTGACCCTTCTAAG-3’, and Ym1 (R) 5’-TCATTACCCTGATAGGCATAGG-3’. Fold changes in mRNA expression were calculated using the ΔΔCT method.[16] The results were normalized to GAPDH mRNA levels in the same samples.
2.9. Establishment of the Nluc -expressing m17.ASC cells
The Nluc sequence was inserted into the pEBMulti/Hyg vector to construct a plasmid encoding Nluc (pEBMulti-Nluc) as previously reported.[17] In brief, the cDNA fragment ofNluc was amplified by PCR and digested using the restriction enzymes KpnI and EcoRV (New England Biolabs, Ipswich, MA, USA). Subsequently, the product was inserted into the multiple cloning site of the pEBMulti-Hyg vector using the same restriction enzymes and Ligation High (Toyobo). m17.ASC cells (5 × 104 cells) were seeded into a 48-well culture plate and cultured for 24 h. The pEBMulti-Nluc plasmid was transfected into m17.ASC cells using Lipofectamine 3000 (InvivoGen). The transfected m17.ASC cells were cultured in DMEM containing 20% FBS for 3 days. The cells were then cultured in media containing 400 μg/mL hygromycin B to selectNluc -expressing m17.ASC (m17.ASC/Nluc) cells. Nlucexpression in cells was confirmed using an Nluc substrate (Nano-Glo Luciferase Assay Reagent, Promega, Madison, WI, USA). m17.ASC/Nluc cells were cultured in a medium containing 400 μg/mL hygromycin.
2.10. Tissue distribution of m17.ASC/Nluc spheroids after intravenous injection in mice
m17.ASC/Nluc spheroids (135 spheroids: approximately 1.5 × 105 cells) or suspended m17.ASC/Nluc cells (1.5 × 105 cells) were intravenously injected into FVB/N mice. Blood and tissues (heart, lung, liver, kidney, and spleen) were collected 24 h after intravenous injection. Tissues were homogenized in a lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 2.5 mM MgCl2, 0.05% (w/v) NP-40, pH 7.4) using a homogenizer (Microtec, Chiba, Japan). Blood and homogenized tissues were centrifuged at 10,000 × g for 15 min at 4 °C to obtain plasma and the supernatant of homogenized tissues, respectively. Subsequently, the relative light units of the plasma and supernatants were measured using the Nano-Glo Luciferase Assay Reagent with an Envision multilabel plate reader (PerkinElmer, Waltham, MA, USA). In addition, m17.ASC/Nluc spheroids (100 spheroids, 24-h incubation: approximately 1.5 × 105 cells) or suspended m17.ASC/Nluc cells (1.5 × 105 cells) were intravenously injected into FVB/N mice. Lungs were collected at 6, 24, 48, 120, and 168 h after intravenous injection and homogenized in the lysis buffer. The homogenates were centrifuged at 10,000 × g for 10 min at 4 °C to obtain the supernatant of the homogenized lungs. The relative light units of the supernatants were measured, as described above.
To image m17.ASC spheroids or suspended m17.ASC cells in the lung after intravenous injection, suspended m17.ASC cells (1 × 105 cells) or m17.ASC spheroids (90 spheroids, 24-h incubation: approximately 1 × 105 cells) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Sigma-Aldrich). Lungs were collected from euthanized FVB/N mice 24 h after intravenous injection and fixed using O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan). Cryosections of the lung were prepared using a Leica CM3050 S cryostat (Leica Microsystems, Nussloch, Germany) and observed under a BZ-9000 microscope (Keyence).
2.11. Effect of m17.ASC spheroids on the LPS-induced inflammatory mouse model
LPS (Sigma-Aldrich) was intraperitoneally administered to FVB/N mice at a dose of 1 mg/kg. The mice were then bred on a hot plate (40 °C) for 1 h. Subsequently, m17.ASC spheroids (850 spheroids, 24-h incubation: approximately 1 × 106 cells) or suspended m17.ASC cells (1 × 106 cells) were intravenously injected in the mice. Blood was collected from each mouse at 6 and 10 h after the injection. Blood was centrifuged at 10,000 × g for 5 min at 4 °C to obtain plasma. The plasma levels of IL-6 and TNF-α were measured using ELISA kits (Thermo Fisher Scientific).
2.12. Statistical analysis
The statistical significance of differences was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons or Student’s t -test for two groups. Differences were considered statistically significant at P <0.05.