Costar 3470

The 3D organoid cultures were performed as described previously [21 (link)]. Freshly isolated AT2 cells (6000/transwell) were mixed with MLg2908 cells (2 × 10⁵/transwell, ATCC CCL-206™, USA) and suspended in a 1:1 mixture of growth factor reduced Matrigel (BD Biosciences) and organoid medium (DMEM/F12 + 2 mM l-Glutamine [MP Biomedicals Europe, Illkirch, France] + 10% Active FBS [A31605-01, Gibco] + 1% ITS [41400-045, Gibco] + 1% Pen/Strep and 10 μM TGF beta inhibitor [Ab120163, Abcam, Cambridge, UK]). The cell mixture was seeded on top of polyester membrane cell culture transwell inserts at 45 μL/transwell (Costar 3470: 0.4 μm pore size, 0.33 cm² area, Corning) and incubated for 30 min to allow the matrix to solidify. We added 600 µL of organoid medium to the bottom of the transwell and replaced half (300 μL) of the medium on alternate days. Cultures were grown at 37 °C and 5% CO₂ for 4–12 days. Then, 10 µM of PKA inhibitor H89 (371963, Millipore, Bedford, MA, USA) was dissolved in the culture medium and applied to cells. For LPS treatment, 1 µg/mL of LPS (L6529, Sigma) was dissolved in the culture medium and applied. H89 and LPS were added to the medium on alternate days.

Transwell inserts (Costar 3470: 0.4 μm pore size, 0.33 cm² area; Corning Costar, USA) were pre-coated with mouse laminin 1 or 5 at 10 μg/cm² (for mouse AT2 cells; Trevigen, USA) for 4 - 6 h at 37°C. Freshly isolated AT2 cells were seeded at 10⁶ cells/cm². The CMM medium (600 μL) was added to the basolateral side of each transwell. The culture medium on the basolateral side was replaced with a serum-free medium 72 h post seeding. Transepithelial resistance (R_T, Ω) and potential difference (V_T, mV) was measured using an epithelial voltohmmeter (EVOM: World Precision Instrument, USA) in 72 h. Cells were cultured for 3 days submerged in culture media and then shifted to the air-liquid interface for another 48 - 72 h.

Sterile 13 mm diameter coverslips (Knittel) were placed in 24-well culture plates. The coverslips were treated with 0.2% gelatin (gelatin from bovine skin—Sigma-Aldrich) diluted in 1 × PBS. PMLECs were seeded in the culture plate (7 × 10⁴ cells/well) in 400 μl of complete DMEM medium, and 1 × 10⁵ peritoneal macrophages (Mφ) were seeded in a Transwell membrane (6.5 mm membrane diameter, 0.4 μm membrane pore, Corning, Costar 3470) without contacting PMLECs on the bottom of the 24-well plate. They only shared the supernatant through the Transwell membrane. After 24 hours, 2.5 × 10⁶ mature iRBCs or RBCs were added over the Mφ and coincubated for 24 hours. After this time, the supernatant between the Mφ and endothelial cells was collected for the quantification of cytokines by ELISA. Subsequently, endothelial cells were coincubated with 1.75 × 10⁶ PbA-iRBCs for the adhesion assay.

Lung DC–T cell cocultures were performed in 24-well flat-bottom culture plates. Briefly, purified DCs (CD11c⁺) from lung were treated with LPS (1 mg/mL) overnight, after which the culture supernatants were removed, and the cells were extensively washed and resuspended in RPMI/10% FCS. Naive CD4⁺ T cells (1 × 10⁶ per well) were then cocultured with the DCs (1 × 10⁵ per well) in the presence of anti-CD3 (2 μg/mL), anti-CD28 (1 μg/mL), IL-4 (20 ng/mL), and TGF-β (2 ng/mL) for 5 days in 1 mL of complete culture medium. Some experiments were performed with DCs and T cells separated in 24-well Transwell cultures (Corning Costar 3470; 0.4 μm pores).

NCI‐H441 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultivated in 25 cm² flasks together with 10 mL of culture medium (RPMI 1640 medium; Pan Biotech, Aidenach, Germany) containing 2.5 μg/mL Na‐pyruvate (Sigma–Aldrich, Taufkirchen, Germany) and 10% charcoal stripped fetal calf serum (Sigma–Aldrich). On day 7, the cell layers reached 80% confluence. For further measurements, the cells were suspended using Trypsin LE (Invitrogen, Darmstadt, Germany) according to the manufacturer's protocol; thereafter, 10⁴ cells were seeded onto 0.33 cm² permeable polyester transwell filter inserts (Costar 3470; Corning, Fisher Scientific, Schwerte, Germany) and were placed in 24‐well plates. The apical compartment was filled with 200 μL and the lower compartment with 500 μL of culture medium. On day 4 after seeding, an ALI was established by removing the medium from the upper compartment. The medium of the lower compartment was replaced by ALI medium (culture medium: +30 nmol/L dexamethasone, 1.72 μmol/L insulin, 68.8 μmol/L transferrin, and 38.7 nmol/L sodium selenite [ITS; Invitrogen]). Medium was exchanged every second day. On days 6–7, the cells were able to maintain a stable ALI. Water transport and I_sc measurements were performed on day 11 after seeding.