6.5 mm transwell

The cell migration was assessed with a transwell migration assay. 1 × 10⁵ transfected cells were added to the upper chamber of the insert and allowed to migrate for 24 h. The cell invasion assays were performed with coating inserts with Matrigel. 1 × 10⁵ cells per well were allowed to infiltrate for 48 h. A 6.5 mm transwell (Corning, Steuben County, NY, USA) with 8.0 µm pore polycarbonate membrane inserts was used to help monitor the cell migration of the transfected cells compared to that of the controls. A medium containing 10% FBS was added to the wells of the plate, followed by the transwell inserts. Afterward, serum-free medium and the associated transfected cells were added to the inner compartment. The cells in the lower chamber were fixed and stained with crystal violet. All the experiments were performed in triplicate.

ASCs were resuspended in cryomilled adipose ECM or reconstituted micronized acellular dermis (Cymetra) at 2 million cells per 50 µL construct and seeded in the top chamber of a 6.5 mm transwell (Corning). After culturing in ASC maintenance media for 48 h (10% FBS, 100 U/mL penicillin, 10 µg/mL streptomycin in DMEM F-12), constructs were transferred into adipogenic induction media (1 μM dexamethasone, 200 μM indomethacin, 500 μM methylisobutylxanthine, 10 μg/mL insulin, 1% penicillin/streptomycin, and 10% FBS in High Glucose DMEM). Cells were differentiated for 7 days in culture on an orbital shaker. For histological analysis, constructs were fixed in 10% formalin overnight, infiltrated with graded sucrose solutions, and embedded in OCT. Samples were cryosectioned at 10 μm sections and stained with hematoxylin and eosin or Oil Red O (0.75% w/v in 36% triethyl phosphate) for lipid accumulation.

Madin–Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection and maintained as previously described.12 Nasal epithelial (obtained by nasal swabbing) and peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers. Informed consent was obtained from all volunteers using protocols and consent forms approved by the Geisel School of Medicine investigational review board (Dartmouth College). A total of four participants (three men and one woman, average age ~59 years) were enrolled in the study. All donors were seropositive to A/California/07/09 (H1N1) virus (mean hemagglutination inhibition titer ~1/32).
Primary NHE cells of passage 2 were grown either in tissue culture flasks (i.e., non‐differentiated cells) or on membrane supports (6·5‐mm Transwell; Corning Inc., Corning, NY, USA) at the air–liquid interface in hormone‐ and growth factor‐supplemented serum‐free medium and fully differentiated 4‐ to 8‐week‐old cultures (~10⁴ cells/insert) were used for the experiments.13 PBMCs were cryopreserved after separation on a Ficoll–Hypaque gradient (Sigma‐Aldrich, St. Louis, MO, USA).
The influenza viruses, A/California/07/09 (H1N1) and A/equine/Tennessee/5/86 (H3N8), were obtained from the World Health Organization's collaborating laboratories.

Migration of the different engineered bMSCs to the targets were conducted using 8.0 μm pore membranes (Corning 6.5 mm Transwell^®). bMSCs or H-bMSCs (1×10⁴ cells/well) were seeded in the upper chamber, and NS, 4T1 cells, or SDF-1α medium were placed into the lower chamber, respectively. After 24 h incubation, the cells on the lower surface of the membrane were fixed with methanol and stained with 0.1% crystal violet for 30 min. Cells that had migrated were observed and counted in three randomly selected microscopic fields (400×) using an Olympus microscope (Olympus, Tokyo, Japan).

HT-29, Caco2, and T84 cells were seeded in 6.5 mm transwells (0.4 μm) (Corning, Corning, NY, USA). After transfection and stimulation with TNF-α, cells were washed with PBS and fixed with cold met-OH for 10 min at 4 °C and subsequently. Permeabilization was performed incubating the monolayer with Triton-X 0.1% in PBS for 5 min at room temperature. Samples were blocked in PBS + BSA 3% for 1.5 h at room temperature. Then, samples were stained with primary antibody rabbit polyclonal Claudin-2 (51-6100, Life Technologies, Carlsbad, CA, USA, dilution 1:100) and diluted in PBS + BSA 3% for 3 h. After washing with PBS, they were stained with secondary antibody chicken anti-Rabbit IgG (H + L) Alexa Fluor 594 (A-21442, Invitrogen, Carlsbad, CA, USA, dilution 1:400) in PBS + BSA 3% for 1 h. ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA) was applied to each sample and then mounted with a glass cover slip. The fluorescence was observed using an Eclipse Ti2 Nikon microscope (Nikon Inc., Melville, NY, USA).

Transwell assays were employed to evaluate the migration captivity. Briefly, 2×10⁵ cells in 200 μL DMEM, supplemented with or without EVs, were seeded onto the upper chamber of 6.5 mm transwells (Corning Incorporated, Corning, NY, US). After incubation for 20 h, the interior U87 or U251 cells of the chamber, which did not penetrate the membrane, were removed. After that, the chambers were fixed with paraformaldehyde and then stained with crystal violet. Finally, the migrating cells were measured in five microscopic fields (200×) (Carl Zeiss, Germany).