3 well silicone insert

TIGK cells were seeded in a 3-well silicone insert (ibidi, Fitchburg, WI, USA) in a well of a 12-well plate. Silicon culture inserts were used to create gaps with a width of 500 µm. Each well was planted with 50,000 cells in 70 μL of the medium. Each 3-well insert created two gaps. When cells reached 100% confluency, they were treated with 1µg/mL mitomycin C (Sigma-Aldrich St. Louis, MO, USA) for 1 h to stop cell proliferation and then washed 3 times with PBS. After the inserts were removed, the culture media was switched to ones with higher concentrations of glucose (24 mM and 48 mM). Images of the gaps were taken at 0 h, 12 h, 24 h, and 48 h post-gap removal. The gap area in pixels was assessed by ImageJ software. Each treatment at each time point had 4 replicates. The percentage of the opened area was calculated based on the initial gap area. There were 3–6 replicates for each culture condition and each time point.

The migrative capacity of MSCs was evaluated using a 3-well silicone insert (for 2-dimensional migration, Ibidi, 80,369) and an 8 μm-pore transwell chamber system (for 3-dimensional migration, Corning, 3422).
In wound healing test, MSCs were seeded into the 3-well culture-insert and cultured until an optically confluent cell monolayer was formed. Cell proliferation was then suppressed by treating the cells with the proliferation inhibitor, mitomycin C (1 µg/mL, Selleck, S8146) for 1 h. The culture-insert was removed to create gaps, followed by replacement of the old medium with fresh one with or without 100 ng/mL human CCL5 (R&D system, 278-RN). Gap closure was monitored by taking pictures at different time points (0, 3, 6, 12, 24 h) under an inverted optical microscope.
In the transwell assay, MSCs were seeded onto the upper chamber (2.0 × 10⁵ per well) placed in a 24-well plate (Corning, 3524), while the lower chamber was loaded with recombinant human CCL5 (100 ng/mL). After 4 h of co-culture, we swabbed the MSCs remaining on the upper surface of the membrane and stained the filter with 0.1% crystal violet (Solarbio, G1064). MSCs that migrated to the lower surface of the membrane were counted under an inverted optical microscope.

Cells (2 × 10⁵ cells/mL) were cultured in 3-well silicone inserts (ibidi GmbH, Gräfelfing, Germany) until monolayer formation. The medium was then replaced with a serum-free medium containing 0.25 mg/mL BSA for 16 h before the inserts were removed and 10 µg/mL galectin-3 or 10 µg/mL BSA was introduced. The gaps were imaged and measured every 12 h until closure.