Intestinal Organoid-Intraepithelial Lymphocyte Coculture

We followed a modified protocol from a previous report (27 (link)). Briefly, intestinal organoids were cultured for 2 days prior to the co-culture with IELs. On the first day of co-culture, SI-IELs collected from the WT mice aged 9-weeks old or 89 weeks old were stained with anti-mouse CD3e-APC-Cy7, anti-mouse CD4-PE-Cy7, and the CD3⁺ (CD3⁺) and CD4⁺ (CD3⁺ CD4⁺) -IELs were sorted using FACSMelody (BD Bioscience). Cultured organoids released from Matrigel were washed and counted. We combined 200 organoids and 2.0 × 10⁵ IELs and centrifuged the samples for 3 min at 200 g. In the control group, the same number of organoids as the co-culture group was centrifuged. The pellet was suspended in 20 μL of Matrigel and placed in 24-well plates. After Matrigel polymerization, 500 μL of the complete medium with 100 U/mL recombinant human IL-2 (Roche), 10 ng/mL mouse IL-7 (Peprotech) and 10 ng/mL mouse IL-15 (Peprotech) were supplemented. The medium was refreshed every 1–2 days. Images of organoids were taken with a BZ-X710 microscope (Keyence).