Transwell with 0.4 μm pore polyester membrane insert

Cells were seeded (4*10⁴ cells/insert) onto 6.5 mm Transwell^® with 0.4 μm Pore Polyester Membrane Inserts (Corning, NY, USA) in 24 well plates. After approximately three days of cell division, the cells were “air-lifted”, a process which involved removing media from the apical chamber and replacing the media in the basal chamber with PneumaCult-ALI Medium (STEMCELL Technologies, BC, Canada). Cells were maintained in ALI conditions for at least three weeks until beating cilia were observed under light microscopy and a high trans epithelial electrical resistance (TEER) was established. A successfully differentiated ALI culture contains basal cells, tight junctions, secretory cells (primarily mucus-secreting goblet cells) and ciliated epithelial cells (Figure S1).

Cells were seeded (4*10⁴ cells/insert) onto 6.5 mm Transwell^® with 0.4 μm Pore Polyester Membrane Inserts (Corning, NY, USA) and differentiated with PneumaCult-ALI Medium (STEMCELL Technologies). A successfully differentiated ALI culture contains basal cells, tight junctions, secretory cells (primarily mucus-secreting goblet cells) and ciliated epithelial cells.

Donor cells were labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher Scientific), 5 μL per ml of cell culture medium for 15 min at 37°C, washed three times. Afterward, cells were seeded in co-culture with acceptor cells in 6-well cell culture plates (1 × 10⁵ MSC cells plus 1 × 10⁵ YFP-positive NALM6 cells) and co-cultured for 24 h. To physical separate the interaction between NALM6-YFP and MSC, MSC and YFP-positive NALM6 cells were plated in the lower and upper chambers of a transwell with 0.4 μm pore polyester membrane insert (Corning). After 24 h, acceptor cells received DiD-labeled components were analyzed by flow cytometry analysis.