Culture inserts 2 well

Migration assay was conducted by Culture-Inserts 2 Well (Ibidi, Germany). In brief, the 2-well silicone inserts were placed at the bottom of a 24-well plate. 70 μl HUVECs suspension was added in both wells of the silicone inserts. After cell adhesion, the silicone inserts were removed, leaving a 500 μm cell-free gap. The monolayer of HUVECs was cultured in DMEM with 2% FBS. The region of migration was photographed at 0 and 48 h after intervention.

Wound healing assay was performed in ibidi chambers (Culture-Inserts 2 Well, ibidi, Germany) in a 24-well plate. The 6 × 10⁵ cells in the volume of 70 microliters were added to each well. After culturing for 24 h in a cell culture incubator, the culture-inserts were taken out. We can saw the scratches at the location where the culture-inserts were removed. The width of the scratch was evaluated visually at 2-time points (0 and 12 h) by light microscopy (CKX41, Olympus, Japan) and measured with Image J software. The experiment was performed in triplicate.

Human coronary artery endothelial cells (HCAEC) were purchased from Cell Systems, Germany. To evaluate initial cell growth, HCEAC were used at passage 3 to 5 and cultured in endothelial cell growth medium (Cell Systems, Germany) containing 10% fetal calf serum (FCS). Cells were seeded with a density of 3 × 10⁵ cells/mL in Culture-Inserts 2 Well (Ibidi, Germany) on a 25 × 75 mm Thermanox™ coverslip (fisher scientific, Germany). According to the manufacturer’s instructions, 70 μL of the cell suspension was added to each well. After 24 h the insert was removed, thereby generating an accurate longitudinal gap between both monolayers of approx. 500 μm. Subsequently, the coverslip was attached to a 0.8 mm sticky-slide I Luer perfusion channel. Using the ibidi pump system (Ibidi GmbH, Germany), endothelial cells were exposed to a constant laminar wall shear stress of 0.15 Pa or 1 Pa in an incubator (5% CO₂, 95% H₂O). Additionally, control samples were kept under static conditions. In the incubator, cell movement was monitored over a period of 15 h using the JuLiTM Life Cell Analyser (NanoEnTek, Korea). Every 15 min a live-cell image was captured. Since this work is intended to serve as a basis for future studies on cell growth under flow conditions, all results presented here are based on wound healing assays under flow.

Culture-inserts 2 well (Ibidi Gmbh, Gräfelfin, Germany) were placed on the center area of the well in 12-well cell culture plates. HaCaT cells were seeded in each well and incubated overnight. With 100% cell confluence, the culture inserts were removed, and the culture medium was changed to a serum-free medium containing 0.4, 0.8, and 1.6 mg/mL of catechu extracts. The culture insert was removed at 0 h. The area of the cell-free gap zone was photographed and defined by drawing the lines. After 48 h, the cell-free gap zone was photographed again and the cells that migrated into the cell-free gap zone acted as an indicator of wound healing [5 (link)]. The cell number was counted manually using the ImageJ software. The results for each group were compared.

Wound-healing assays were performed using IBIDI^® culture-Inserts 2 Well for self-insertion. Each insert was placed in a well of a 12-well plate. MSC (5 × 10⁴) in 200 μl DMEM were seeded in a well of the insert. The inserts were then removed, and DMEM without or with TNF-α at a concentration ranging from 10 to 100 ng/ml was added. After 12 h of migration, the migratory area was calculated using ImagePro Plus 6.0. The percentage of the migratory area was defined as the ratio of the MSC migratory area at 12 h to the primary wound area created by the insert at 0 h.

For left‐to‐right migration model, hADSCs and MG63 spheroids were separately seeded on the left and right side of Culture‐Inserts 2 Well (ibidi). Briefly, hADSCs were labeled with PKH67 (sigma) according to the manufacturer's instructions. Then, H@C@D nanorods were added into the PKH67‐labeled hADSCs suspension and incubated for 30 min at 37 °C with 5% CO₂ and 95% humidity. After that, the nADSCs were rinsed with PBS for three times to wash off the unlinked nanorods. And the MG63 single cell suspension was labeled with PKH26 and then added to 24‐microwell plate to generate the spheroids. Then the PKH26‐labeled MG63 spheroid were dispersed in Matrigel (1 mg mL⁻¹) and seeded on the right side of Culture‐Inserts while PKH67‐labeled H@C@D‐loaded hADSCs were seeded on the left. After 24 h incubation, the Culture‐Inserts were removed and the migration state was observed with CLSM at specific time point. For top‐to‐bottom migration model, the petri dish was first coated with Matrigel (2 mg mL⁻¹) at 37 °C. Then the PKH26‐labeled MG63 spheroids were dispersed in Matrigel (1 mg mL⁻¹) and seeded on the coated plate. After culturing at 37 °C with 5% CO₂ and 95% humidity for 6 h, the PKH67‐labeled H@C@D‐loaded hADSCs were seeded on the top of the Matrigel layer. The migration state was observed with CLSM at specific time point.