Extended Data Fig. 3: Cytokine supplementation and cryopreservation of intestinal ALI organoids.

a, FACS-based tSNE plots depicting time course abundance of EPCAM⁺ and CD45⁺ cells (top) and CD4⁺ and CD8⁺ T cells (bottom) as a percentage of total live single ileal organoid cells with or without addition of IL-2 and IL-7, representative experiment of n = 3 biological replicates. b, Organoids grown for 14 days (control) have similar percentages of epithelium and immune components as organoids grown for 5 days, frozen in-gel at −80 °C, cryorecovered, and replated for the indicated durations. c-d, ALI organoids demonstrate persistent growth after being frozen in-gel at −80 °C, cryorecovered and replated (c, arrows), with maintenance of epithelial protrusions by H&E (d). Numerous air bubbles in the collagen are present on initial plating post-cryorecovery and progressively disappear with culture. (b-d) depict representative experiments from n = 4 biological replicates. Scale bar is 5 mm for (c) and 100 µm for (d).