3d matrigel

For passaging the alveolar organoids, 3D Matrigel was dissolved by adding 150 μL of dispase (Corning) to the apical chamber of the inserts and incubating for 1 hour at 37°C followed by 0.25% trypsin/EDTA (Invitrogen) for 5 minutes at 37°C. The reaction was quenched with 10% FBS DMEM/F12. Pellets were resuspended in staining buffer, and single-cell suspensions were stained using antibodies against EPCAM (9C4), HTII-280 (Terrace Biotech), and DAPI (Sigma) for viability determination. Labeled cells were sorted for EPCAM⁺ using a FACSAria Fusion; HTII-280 expression status was used for analysis. A total of 5 × 10³ EPCAM⁺ viable cells were mixed with 5 × 10⁴ MRC5 fibroblasts (ATCC CCL-171) and resuspended in a 50:50 (v/v) ratio of ice-cold 3D Matrigel (Corning) and CK+DCI medium. Next, 100 μL of the suspension was seeded on a 0.4 mm–pore cell culture insert in a 24-well supported format (Corning). After polymerization of Matrigel, 500 μL of CK+DCI medium was added to the bottom well. Medium was supplemented with 10 μM Y-27632 for the first 48 hours. Medium was changed every other day.

CD31^–CD45^–EPCAM⁺HTII-280⁺ alveolar epithelial cells were resuspended in CK+DCI medium, 3D medium (DMEM/F12 supplemented with 10% FBS, penicillin/streptomycin 1,000 U/mL, 1 mM HEPES, 1 mM l-glutamate, and insulin/transferrin/selenium from Sigma), or SAGM as shown at a density of 5 × 10³ cells/50 μL and then mixed with 3D Matrigel (Corning) containing MRC5 fibroblasts (ATCC CCL-171) at a density of 50 × 10³ cells/50 μL. A total of 100 μL of the suspension was seeded on a 0.4 mm–pore cell culture insert in a 24-well supported format (Corning). After polymerization of Matrigel, 500 μL of CK+DCI medium was added to the bottom chamber. Medium was supplemented with 10 μM Y-27632 for the first 48 hours. Medium was changed every other day. Cultures were maintained at 37°C in a humidified incubator (5% CO₂).