Isolation and Culture of Primary Intestinal Epithelial Cells

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.