Dmi8 s microscope

Cell-embedded matrices were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 0.01% of 2-mercaptoethanol, and 1% penicillin/streptomycin at 37 °C, 5% CO₂, and 95% humidity. Cell culture media and supplements were purchased from Invitrogen, CA, USA.
THP-1 cells were differentiated into uncommitted macrophages using 300 nM phorbol 12-myristate 13-acetate (PMA; Merck KGaA, Germany)) in RPMI-1640 cell culture media without FBS supplement, according to the established protocol^{18 ,40} . After 6 h, differentiation media was removed, cell-embedded matrices were washed with PBS, and rested for 24 h in RPMI-1640 without FBS supplement nor PMA. Afterwards, cells were activated for 48 h into two different macrophages subtypes M2_IL-4/IL-13 by treatment with 20 ng/ml interleukin 4 (IL-4; Biolegend, CA, USA) and 20 ng/ml interleukin 13 (IL-13; Biolegend, USA), or into M2_IL-10 by adding 20 ng/ml interleukin 10 (IL-10; Biolegend, USA).
After successful cell activation, cells were imaged in a bright-field mode by a DMi8 S microscope using ×20 long distance objective (NA 0.4; Leica, Germany). Cell area and cell aspect ratio (cell length/cell width) were analyzed using an automated image analysis toolbox^{38 (link)}. At least 100 cells were analyzed. The experiment was performed at least in triplicates.

HAECs and hASMCs were expanded as before. Prior to harvesting, medium was aspirated from the cells, and cells were incubated for 30 min with 10 μM CellTracker Green CMFDA and Orange CMTMR (Invitrogen), respectively, for 30 min. The cells were then washed with PBS twice and harvested. Both HAECs and HASMCs were resuspended in smooth muscle cell growth medium and combined 1:1 for a total of 80.000 cells mL⁻¹. Five hundred microliters of mixed‐cell suspension was seeded on each of the coverslip, which corresponds to approximately 20.000 hAECs and 20.000 hASMCS cm⁻². After 24 h of culture, the cells were visualized before and after fixation in 3.7% formaldehyde and counterstaining with DAPI using a Leica DMI8s microscope.

Solution‐cast films on 14‐mm glass coverslips were secured in an adapted Transwell as described before67, 68 and sterilized under UV for 15 min. Endothelial cells were seeded at a density of 40.000 cells cm⁻² and smooth muscle cells at 25.000 cm⁻². Five hundred microliters of cell‐suspension was used inside the inserts, and 1 mL of appropriate growth medium was added outside the insert after establishing the inserts did not leak immediately. Cells were cultured for 24 h at 37 °C and 5% CO₂, after which non‐adherent cells were aspirated and the surfaces were washed with PBS with subsequent fixation in 3.7% formaldehyde. The actin‐cytoskeleton was stained with ATTO488 conjugated phalloidin, and nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Samples were visualized using a Leica DMi8s microscope. Furthermore, the surface covered by cells was determined by binarizing fluorescence micrographs of the phalloidin‐stained samples into background and cells in ImageJ (NIH, version 1.48). The percentage of foreground pixels was used as a measure for the area occupied by cells. Three images taken with a 10x objective were measured per condition.