Millicell 24 cell culture insert plate system

Contact and noncontact coculture models were adopted from our previous investigation [5 (link)] which was demonstrated in Figure 1(a). According to previous descriptions, macrophages were cocultured with VSMCs in Millicell-24 Cell Culture Insert Plate system with polyethylene terephthalate membranes (Millipore) [21 (link), 22 (link)]. For the contact coculture model, macrophages (4 × 10⁴/cm²) were seeded to the basal surface of the insert. Then, the insert was reverted into 6-well culture plates. VSMCs (4 × 10⁴/cm²) were seeded to the apical surface of the insert. Then, the system was incubated in RPMI-1640 medium supplemented with 10% FBS, 2 mmol/L glutamine, and antibiotics mix for 72 h at 37°C. For the noncontact model, macrophages (4 × 10⁴/cm²) were seeded to the bottom of the well.

Two co-culture models, contact and non-contact, were employed in this study^{11 (link)} (Fig. 6C). Macrophages were co-cultured with VSMCs using a Millicell-24 Cell Culture Insert Plate system equipped with polyethylene terephthalate membranes (Millipore). In the contact co-culture model, macrophages were first seeded onto the basal surface of a cell culture insert at a density of 4 × 10⁴ cells/cm². Using RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mmol/L glutamine (Gibco), and a combination of penicillin and streptomycin (Gibco), the macrophages were allowed to adhere overnight at 37 °C in a humidified atmosphere containing 5% CO₂. Following this, the insert was carefully inverted, and VSMCs were seeded onto its apical surface at a density of 4 × 10⁴ cells/cm² allowing them to adhere overnight under the same conditions. After the insert was carefully re-inverted, the entire assembly was then incubated for 72 hours in the supplemented RPMI-1640 medium. In contrast, for the non-contact co-culture model, VSMCs were seeded directly onto the bottom of the culture well, ensuring that while both cell types were immersed in the same medium, direct physical interaction was prevented. The rest of the procedure mirrored the contact model, with cells maintained in the supplemented RPMI-1640 medium for the designated period.

Both contact and non-contact co-culturing systems were used in this study. Primary VSMCs and treated macrophages were co-cultured with a Millicell-24 Cell Culture Insert Plate system with polyethylene terephthalate (PET) membranes (Millipore, Billerica, MA, USA) according to several previous descriptions (10 (link), 19 (link)), which is also demonstrated in Figure 4A. For the contact model, macrophages were seeded to the basal surface of the insert at a density of 4 × 10⁴/cm² and incubated overnight for adherence. The insert was then reverted into 6-well-culture plates and incubated in RPMI-1640 medium supplemented with 10% FBS, 2 mmol/L glutamine, and antibiotics mix at 37°C. VSMCs were seeded to the apical surface of the insert density of 4 × 10⁴/cm² and incubated overnight for adherence. The system was then incubated in culture plates in RPMI-1640 medium supplemented with 10% FBS, 2mmol/L glutamine, and antibiotics mix at 37°C for 72 h. For the non-contact model, macrophages were seeded to the bottom of the well-instead of the basal surface of the insert.