Juli stage system

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).

HUVEC cells were cultured on 96-well plates and incubated until reaching 70% confluence. After culturing for 24 h, the confluent cells were wounded by scratching with a manufacturer provided scratcher (NanoEntek, Seoul, Korea), and the wells were rinsed with 100 μL of fresh medium. Afterwards, medium was replaced with the same volume of fresh medium (control) or medium including compounds at various concentrations (10 + 90 μL, final concentration of compounds 0.1 and 0.5 μmol/mL). The plates were incubated up to 36 h at 37 °C (5% CO₂). In this period, migration of cells was monitored using the JuLI™ Stage system which is a Real-Time CHR (Cell History Recorder, NanoEntek, Seoul, Korea) designed for live cell imaging and analysis. The images of cells migration were acquired continuously every 10 min (high-sensitivity monochrome CCD (Sony sensor 2/3”, Tokyo, Japan)). The images were analyzed by dedicated software (NanoEntek, Seoul, Korea), and the width of the scratch area was measured. The results are presented as mean ± SD, n = 4–8. The coefficients of variation for the applied method were determined (CV = 13.6–16.0%, depending on the time point, n = 8)

The migration of cells under different chemokine gradients was analyzed using ibiTreat 2D “mu (µ)‐Slide” Chemotaxis system (Ibidi GmbH, Martinsried, Germany).^[28] Before the chemotaxis assay, the surface of the slide for was coated with fibronectin (F2006, 300 µg mL⁻¹, Sigma) for 1 h for cells attachment. After washing with PBS, 6 µL of cells (3 × 10⁶ cells mL⁻¹) were loaded into the central observation channel and incubated at 37 °C for 45 min to allow attachment. Gradients of CXCL12 were generated according to the manufacturer's instructions. For the migration track analysis, live cell imaging was recorded for indicated periods and intervals on a JuLi stage system (NanoEnTek, Seoul, Korea) operating on a CO₂ incubator. The tracking of migrating individual cells was achieved using the Manual Tracking plugin (Institut Curie, Orsay, France) in ImageJ software (NIH, Bethesda, MA). Chemotaxis plots were obtained with the Chemotaxis and Migration plugin from Ibidi. To analyze the actin dynamics of live cells during chemotaxis, the cells were stained with SiR‐Actin, a fluorogenic, cell permeable and highly specific probe for F‐actin (SC001, 1 × 10⁻⁶m, Spirochrome, Stein am Rhein, Switzerland) for 1 h before seeding onto “mu‐Slide” and time‐lapse images were obtained using confocal microscope (Nikon) with a 60× oil objective and a temperature‐controlled chamber (37 °C, 5% CO₂).