Isolation and Characterization of ADMSCs

Adipose-derived MSCs (ADMSCs) were obtained from adipose tissue from three healthy human donors as described previously (Ganbold et al., 2019 (link)). All experimental procedures in this study were approved by the Institutional Review Board of the School of Dentistry, Seoul National University (IRB No. S-D20150019). In brief, lipid tissue was collected from liposuction specimens, digested with 0.1% collagenase I (Gibco, Carlsbad, CA) in Hanks’ balanced salt solution (HyClone Laboratories, Logan, UT), and passed through a 100-µm strainer (BD Falcon, Franklin Lakes, NJ). Cells were resuspended in high-glucose Dulbecco’s modified Eagle’s medium (HyClone Laboratories) containing 10% fetal bovine serum (HyClone Laboratories). All cultures were maintained in a humidified incubator at 37°C and 5% CO₂. Cells were passaged at ∼70% confluence, and cells at passages 3 to 7 were used. To generate MiBs, ADMSCs were seeded on AggreWell™400 plates (Stem Cell Technology, Cambridge, MA) and cultured for the 1, 2, 3, or 7 days to form MSC spheroids. To collect spheroids of uniform size, spheroids were passed through double-stacked cell strainers with pore sizes of 70 μm and 300 μm. MiBs were imaged using the JuLi stage system (NanoEnTek, Seoul, Korea), and MiB diameter was measured using ImageJ software (National Institutes of Health, version 1.53j, https://imagej.nih.gov/ij/download.html).