Gastrin 1

Mouse lungs were removed, flushed, cut into 5-mm³ pieces and incubated with digestion buffer containing 1.0 mg ml⁻¹ collagenase I (Gibco, 17100–017) and 0.5 mg ml⁻¹ collagenase IV (Gibco, 17104–019) in DMEM/F12 (Gibco, C11330500BT) in gentleMACS C tubes (Miltenyi Biotec, 130-096-334). After dissociation by the gentleMACS dissociator, samples were filtered through 70-μm filters, collected by centrifugation and resuspended in ice-cold Matrigel (BD, 354230) at a ratio of 1:20 (vol:vol). The basic culture medium for mouse lung organoids was slightly modified from a previous report^{43 (link)}, where DMEM/F12 was supplemented with penicillin/streptomycin (Gibco, 15140–122), 2 mM GlutaMAX (Peprotech, 35050–061), 1× B27 (Gibco, A3582801), 1× N2 (Gibco, 17502048), 10 nM gastrin I (Peprotech, 1003377), 1 mM N-acetylcysteine (Sigma, A9165) and 10 mM nicotinamide (Sigma, N0636). The following growth factors were used: 50 ng ml⁻¹ mouse recombinant epidermal growth factor (Peprotech, AF-100-15-1000), 100 ng ml⁻¹ mouse recombinant noggin (Peprotech, 120-10C-250), 500 ng ml⁻¹ mouse recombinant FGF10 (Peprotech, 100-26-1000), 125 ng ml⁻¹ R-spondin-1 (Peprotech, 120-38-1000), 10% Wnt-3A conditioned medium and 500 nM A83–01 (Peprotech, 9094360). The medium was changed every 3 d. Organoids were passaged by mechanical dissociation in TrypLE (Gibco, 12605–028) every 5–7 d.

Human organoids were derived from young (15–21 years old) and aged (>55 years old) patient stomach tissue collected from sleeve gastrectomies (IRB Protocol number 2015–4869) as previously described [16 (link)]. Briefly, stomachs were washed with DPBS, and mucosa was collected and cut into small fragments. Tissue fragments were incubated at 37°C supplemented with oxygen flow in DMEM/F12 with 1mg/mL Collagenase and 2mg/mL Bovine Serum Albumin. Glands were filtered and centrifuged at 65 × g for embedding into Matrigel. Human gastric organoid growth media was changed every 4 days (Advanced DMEM/F12 medium (Thermo fisher), 50% Wnt conditioned medium, 20% R-spondin conditioned medium, 1X Penicillin/Streptomycin, 1X Amphotericin/Gentamicin, 1X Kanamycin, 1X B27, 1X N2, 1mM N-Acetylcysteine, 10mM Nicotinamide, supplemented with growth factors including bone morphogenetic protein inhibitor, (Noggin, PeproTech), Gastrin I (Tocris), Epidermal grow factor (EGF, PeproTech), Y-27632 (Sigma), and Fibroblast growth factor 10 (FGF-10, PeproTech)) [16 (link)]. Human organoids were cultured at 37°C for 7 days prior to transplantation into NOD scid gamma (NSG) mice.