cAT-MSCs were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Welgene, Gyeongsan-si, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco, Billings, MT, United States) and 1% penicillin–streptomycin (PS; PAN-Biotech, Aidenbach, Germany). The canine macrophage cell line DH82 (ATCC number: CRL-10389; Manassas, VA, United States) was cultured in DMEM supplemented with 10% FBS and 1% PS. All cells were incubated at 37°C in a 5% CO₂ environment. The culture medium was replaced every 2–3 days, and cells were subcultured upon reaching 70–80% confluency.

Canine adipose tissue was collected from three healthy adult female dogs that had undergone ovariohysterectomy at the Seoul National University Veterinary Medical Teaching Hospital (SNU VMTH). This procedure was conducted following a protocol approved by the Institutional Animal Care and Use Committee of SNU (protocol no. SNU-220818-1). MSC isolation and culture were performed as previously described (19). Adipose tissue samples were washed with Dulbecco’s phosphate-buffered saline (DPBS; Welgene) supplemented with 1% PS. The tissue was then minced using sterilized scissors, treated with 0.1% collagenase type IA (Gibco), and incubated at 37°C for 1 h. After incubation, the samples were neutralized with DMEM supplemented with 10% FBS and centrifuged at 1,200 × g for 5 min. The resulting pellet was passed through a 70-μm cell strainer (Thermo Fisher Scientific, Waltham, MA, United States) and centrifuged again at 1,200 × g for 10 min. To remove any remaining blood cells, the pellet was resuspended in RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, United States), incubated at room temperature for 5 min, washed with DPBS, and centrifuged at 1,200 × g for 5 min. The supernatant was discarded, and the pellet was resuspended in DMEM containing 10% FBS and 1% PS. The cells were seeded in 100 mm cell culture dishes at a density of 3,000 cells/cm² and incubated at 37°C in 5% CO₂ environment. The medium was replaced every 2–3 days, and cells were subcultured upon reaching 70–80% confluence, exhibiting a fibroblast-like morphology.

To evaluate their differentiation capacity, cAT-MSCs were cultured in media specific for adipogenesis, osteogenesis, and chondrogenesis (StemPro Differentiation Kits; Gibco). Differentiation into adipocytes, osteocytes, and chondrocytes was confirmed by staining the cells with Oil Red O, 1% Alizarin Red, and Alcian Blue (Sigma-Aldrich), respectively. Surface marker expression was analyzed by flow cytometry using a FACSAria II system (BD Biosciences). Cells were stained with monoclonal antibodies against CD44-FITC, CD45-FITC, CD29-PE, CD73-PE, and CD90-PE (BD Biosciences). Fluorescence data were analyzed using FlowJo software (Tree Star, Woodburn, OR, United States).

cAT-MSCs were seeded at a density of 2 × 10⁶ cells per well in a Stem FIT 3D cell culture dish (c253000, MicroFIT, Inc., Ha-Nam, Republic of Korea). The cells were incubated in DMEM containing 20% FBS and 1% PS in a 5% CO₂ environment at 37°C. Spheroid formation was monitored every 12 h for 48 h under an inverted microscope (Olympus CKX53; Olympus, Tokyo, Japan) at 4 × magnification. Spheroid areas were measured using ImageJ software (National Institutes of Health, United States, http://imagej.nih.gov/ij/).

After 48 h of culture, spheroids were collected by gentle pipetting, excluding any broken spheroids. The spheroids were washed three times with PBS and fixed with neutral-buffered 10% formalin overnight at 4°C. The fixed spheroids were embedded in Histogel (Epredia, Kalamazoo, MI, United States), followed by paraffin embedding, sectioning, and staining with hematoxylin (Thermo Fisher Scientific) and Eosin Y (Thermo Fisher Scientific).

After fixation in 2.4 sections, the fixed spheroids were washed three times with 0.1% Triton X (Sigma-Aldrich) in DPBS (0.1% PBSTX) and permeabilized with 0.5% Triton X in DPBS (0.5% PBSTX) for 2 h at room temperature (RT). Spheroids were then washed three times with 0.1% PBSTX and blocked with 2% bovine serum albumin (Sigma-Aldrich) in 0.1% PBSTX for 1 h at RT. The spheroids were incubated overnight at 4°C with OCT4 rabbit pAb (1:100; ABclonal Technology, Boston, MA, United States), and SOX2 rabbit pAb (1:100; ABclonal Technology). After washing, the spheroids were incubated with goat anti-rabbit antibody conjugated with Alexa Fluor 488 (1:1000; Thermo Fisher Scientific) After incubation, the spheroids were washed three times with DPBS and mounted in an antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, United States). The spheroids were analyzed using a confocal laser scanning microscope (CLSM) (LSM 710; Zeiss, Oberkochen, Germany).

Protein extraction from 2D- and 3D-cultured cAT-MSCs was performed using the PRO-PREP Extraction Solution (iNtRON Biotechnology) according to the manufacturer’s instructions. Protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, United States). For protein expression analysis, 10 μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, United States). Membranes were blocked with 5% skim milk in Tris-buffered saline and incubated overnight at 4°C with primary antibodies against SOX2 (1:1000; ABclonal Technology,), OCT4 (1:1000; ABclonal Technology), and β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, United States). After washing, membranes were incubated with either goat anti-mouse or goat anti-rabbit horseradish peroxidase-labeled secondary antibodies (Bethyl Laboratories, Montgomery, TX, United States, and Enzo Life Sciences) at RT for 1 h. Immunoreactive bands were visualized by chemiluminescence (Advansta, San Jose, CA, United States) and imaged using an ImageQuant Las4000 mini (GE Healthcare Life Sciences, Chicago, IL, United States). Protein levels were normalized to β-actin.

Total RNA from 2D- and 3D-cultured cAT-MSCs was extracted using an Easy-Blue Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Republic of Korea) according to the manufacturer’s instructions. RNA concentration and purity were measured using a spectrometer (Implen, Westlake Village, CA, United States). cDNA synthesis was performed using 1 μg of total RNA with the CellScript All-in-One 5 × 1st cDNA Strand Synthesis Master Mix (CellSafe, Yong-In, Republic of Korea). qRT-PCR was carried out in triplicate using 10 μL AMPIGENE qPCR Green Mix Hi-ROX with SYBR Green dye (Enzo Life Sciences, Farmingdale, NY, United States), 1 μL of cDNA, and 400 nM of forward and reverse primers (BIONICS, Seoul, Republic of Korea). Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are listed in Table 1.

The concentration of PGE2 in each cell culture supernatant was measured using a PGE2 Enzyme-Linked Immunosorbent Assay Kit (Enzo Life Sciences, Farmingdale, NY, United States) following the manufacturer’s instructions.

To compare the anti-inflammatory effects of conditioned media (CM) from 2D- and 3D-cultured cAT-MSCs, as well as dexamethasone, CM was collected from 2D cultures when the cells reached 80% confluence and from 3D cultures after 48 h. The media were centrifuged at 3000 rpm for 10 min at 4°C to remove cell debris and then stored at −70°C until use. DH82 cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/well and treated with 200 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich, United States) for 24 h. Afterward, DH82 cells were treated with CM diluted 1:1 with DMEM containing 10% FBS and 1% PS, or with dexamethasone. After 48 h, macrophage polarization was assessed by flow cytometry using antibodies against CD86 (clone GL-1, APC conjugate; Tonbo Biosciences) and CD206 (clone C068C2, Alexa 488 conjugate; BioLegend). Cells were analyzed using a BD LSR flow cytometer, and data were processed using FlowJo software (Tree Star, Woodburn, OR, United States). Differences in the expression of TNFα, IL-1β, and IL-6 among the groups were evaluated by qRT-PCR.

Each experiment was performed at least three times. GraphPad Prism (version 9.3.1) (GraphPad Software, San Diego, CA, United States; RRID:SCR_002798) was used for statistical analyses. One-way analysis of variance (ANOVA) was used to analyze the data, and Tukey’s multiple comparison test was performed. The findings are shown as mean plus standard deviation (SD). Differences were considered statistically significant at p < 0.05.

We confirmed that the cells used in the experiment were cAT-MSCs by assessing their differentiation potential and surface marker expression. The cells successfully differentiated into adipocytes, osteoblasts, and chondrocytes, as indicated by positive staining with Oil Red O, Alcian Blue, and Alizarin Red S, respectively (Figure 1B). Additionally, flow cytometry analysis showed that the cells expressed the stem cell markers CD44, CD29, CD73, and CD90, while lacking expression of the hematopoietic marker CD45 (Figure 1C). These results confirm that the cells used in the experiments were indeed cAT-MSCs.

We aimed to form spheroids using cAT-MSCs. For this purpose, we cultured 2 × 10⁶ stem cells in a Stem FIT 3D cell culture dish and imaged them at 12 h intervals (Figure 2A). The results showed that stem cells began to aggregate into spheroids as early as 12 h, with the size of the spheroids significantly decreasing after 36 h (Figure 2B). Spheroids were harvested at 48 h, sectioned, and stained with H&E. As shown in Figure 2C, the stem cells within the spheroids aggregated well. Additionally, to determine whether hypoxic conditions were induced during spheroid formation in the 3D culture, we used pimonidazole staining and confirmed the presence of hypoxic conditions (indicated by red staining) (Figure 2D).

Previous studies have reported that the expression of SOX2 and OCT4 in MSCs increases under hypoxic conditions (20, 21). To determine whether SOX2 and OCT4 expressions are similarly elevated under hypoxic conditions in cAT-MSC spheroids, we performed fluorescence staining and confirmed SOX2 and OCT4 expression using confocal imaging (Figures 3A,B). Furthermore, compared to 2D cultures, the expression of SOX2 and OCT4 was significantly increased in cAT-MSC spheroids, as verified by western blotting (Figures 3C,D).

Given that the overexpression of SOX2 and OCT4 in MSCs has been associated with enhanced anti-inflammatory effects (22), we examined the gene expression of inflammation-related factors in spheroid-cultured cAT-MSCs. The mRNA levels of TGFβ1, HIF1α, TSG-6, and COX-2 were found to be elevated in 3D cultures compared to the 2D cultures (Figure 4A). Similarly, the mRNA levels of the inflammatory cytokines IL-1β, IL-6, and IL-10 were also increased in 3D cultures compared to the 2D cultures (Figure 4B). Additionally, the angiogenesis and tissue regeneration factors VEGF-A and bFGF exhibited higher mRNA levels in 3D cultures compared to the 2D cultures (Figure 4C). Finally, there was an increase in PGE2 protein expression in 3D cultures compared to the 2D cultures (Figure 4D).

As shown in Figure 4, the gene expression of inflammation-related factors was higher in 3D-cultured cAT-MSC compared to 2D cultured cAT-MSC. We, therefore, investigated the immunomodulatory effects of CM from 3D-cultured cAT-MSCs on LPS-induced macrophages (DH82 cells). DH82 cells were initially treated with 200 ng/mL LPS for 24 h, followed by additional 48-h incubation with a 1:1 mixture of the original culture medium and CM. We then evaluated macrophage polarization and the expression of pro-inflammatory cytokines.

The results showed that M1 polarization was significantly reduced in the group treated with CM from 3D-cultured cAT-MSC compared to the group treated with LPS alone. However, no significant changes were observed in M2 polarization. Interestingly, there were no significant changes in M1 polarization when comparing the group treated with CM from 3D-cultured cAT-MSCs to the group treated with CM from 2D-cultured cAT-MSCs (Figures 5A,B). Nevertheless, a significant decrease in the expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-6 was observed in the group treated with CM from 3D-cultured cAT-MSCs compared to both the LPS alone-treated group, and the group treated with CM from 2D-cultured cAT-MSC (Figure 5C).