Ax 511

Primary epithelial cells were isolated as described previously [82 (link)] with slight modifications. Briefly, small intestines were harvested from 4 mice, opened longitudinally, and washed extensively with RPMI1640 medium (Nacalai, Kyoto, Japan) after mesentery, fats, Peyer’s patches, and luminal content were removed. The intestines were cut into pieces and shaken gently in RPMI-1640 containing EDTA (2 mM) and 10% fetal bovine serum (FBS) (Equitech-Bio, Kerrville, TX, USA). The tissue preparations were filtered with 70-μm mesh filters. Using 25%, 40%, and 75% Percoll (GE Healthcare Life Sciences, Chicago, IL, USA), the whole cells were spun in a centrifuge (AX-511) (Tomy, Tokyo, Japan) at 780× g for 20 min and IECs were obtained from the interface between the 25% and 40% layers. After verification of their expression of epithelial marker, the IECs were seeded in 12-well culture plates (Corning, Glendale, AZ, USA) at 4 × 10⁵ cells/mL in RPMI1640 containing penicillin/streptomycin and EV-depleted FBS and incubated at 37 °C with 5% CO₂ for 12 h. Then, the cells were treated with the indicated concentrations of EVs for 24 h after which RNA was extracted for further analysis.

Epithelial cells were isolated from mouse lung tissues by using a mechanical dissociation with sterile sieve meshes followed by a discontinuous density-gradient centrifugation using Percoll (GE Healthcare Life Sciences, Chicago, IL, USA) in accordance with previous studies [17 (link),18 (link)] with some modifications. Three Percoll solutions (25%, 40%, and 75%) were prepared, the cells from lung tissues were resuspended in 40% solution, and three distinct gradients were carefully placed in the order of 25%–40%–75% from top to bottom in a 15 mL centrifuge tube (Corning, Glendale, AZ, USA). The cells were spun at 780× g for 20 min in a centrifuge (AX-511) (Tomy, Tokyo, Japan) at the setting of minimal acceleration and deceleration. The epithelial cells were centered at an interface between 25% and 40% Percoll solution and were then taken and washed extensively with RPMI1640 (Nacalai) containing 10% FBS (Equitech-Bio) and penicillin/streptomycin (Nacalai).

SHED and SHED-CM were prepared as previously described^{26 (link)}. The pulp was gently removed and digested for 1 h at 37 °C (SDminiN, Taitec, Koshigaya, Japan) in a solution containing collagenase type I (3 mg/ml) (Fujifilm Wako, Tokyo, Japan) and dispase (4 mg/ml) (Fujifilm Wako). Single-cell suspensions were plated on culture dishes in DMEM (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and then incubated at 37 °C in 5% CO₂ (Forma 310 Direct-Heat CO₂ Incubator, Thermo Fisher Scientific). The human skin fibroblast line, derived from a 36-year-old individual, was obtained at passage 12 from the Health Science Research Resources Bank (Osaka, Japan). SHED (passage 9), and Fibro (passage 13) for CM preparation were seeded at 1 × 10⁵ cells per dish. Cells at 70–80% confluence were washed with PBS and serum-free DMEM, followed by replacement with serum-free DMEM. Media were incubated for 48 h at 37 °C in a humidified atmosphere of 5% CO₂ (Forma 310 Direct-Heat CO₂ Incubator), then collected and centrifuged for 10 min at 2000 × g at 4 °C (AX-511, Tomy Seiko, Tokyo, Japan). Since the precipitates contained cell debris, we carefully collected the conditioned medium without nearing the precipitates. We adjusted the protein concentration of each CM to 3 µg/ml in serum-free DMEM.