5% saponin for 15 minutes with repeated pipetting. A total of 10 μL of 1:10 dilution selleck rows were plated on horse serum agar plates. For heat inactivation, bacteria were kept at 56°C for 1 hour. To test inflammatory stimuli, organoids were incubated with medium containing the following substances in the final concentration: lipopolysaccharide (LPS) from Escherichia coli (1
μg/mL; Invivogen), recombinant human tumor necrosis factor (TNF)α (10 ng/mL; BD Pharmingen), recombinant human interleukin (IL)1β (100 ng/mL; Sigma-Aldrich), CpG oligodeoxynucleotide (ODN) 1668 (1 μg/mL; Enzo), and flagellin from Salmonella typhimurium (100 ng/mL; Invivogen). The reader is referred to the Supplementary Materials and Methods section for fluorescence-activated cell sorting, polymerase chain reaction (PCR) and microarray, cell viability assay, karyotyping, histology, and imaging. To generate a culture system for human gastric epithelium, we isolated gastric glands from human gastric corpus tissue AG-14699 (Figure 1A) and observed their growth under different culture conditions. We started from the conditions for mouse gastric epithelium, 4
containing EGF, noggin, R-spondin1, Wnt, FGF10, and gastrin (ENRWFG). Isolated glands from human donors could form organoids in these conditions with very low efficiency and with a limited lifespan in vitro. We then tested a panel of growth factors and inhibitors for organoid-forming efficiency, phenotype of the organoids, and longevity of the human gastric cultures. TGFβ inhibitor,
p38 inhibitor, GSK2β inhibitor, and PGE2 were chosen because of the relevance of these respective pathways in cancer. IGF is expressed in normal gastric tissue.10 Nicotinamide suppresses sirtuin activity.19 Similar to human intestine,17 nicotinamide increased the number of human gastric organoids formed (Figure 1B and Supplementary Figure 1A). It therefore was included in the subsequent culture condition. IGF, p38 inhibitor, GSK3β inhibitor, and TGFβ inhibitor all induced budding structures in a concentration-dependent manner ( Supplementary Figure 1B) and had a positive effect on the lifespan of the organoids ( Figure 1C). PGE2 induced growth of large cysts and also prolonged the lifespan of the cultures. Addition of TGFβ inhibitor increased click here the lifespan to a maximum of half a year ( Figure 1C), whereas all other factors had no such effect. We therefore only added TGFβ inhibitor to the ENRWFG culture medium. To analyze the importance of the single factors, we then withdrew each of the components from the medium. Without EGF, noggin, R-spondin1, or Wnt, organoid formation was strongly reduced and cultures deteriorated within 1–3 weeks ( Figure 1D and Supplementary Figure 1C). Removal of FGF10, gastrin, or TGFβ inhibitor allowed growth for 10–20 weeks. Removal of nicotinamide increased the lifespan of the cultures ( Figure 1D).