For this, we plotted the cells using forward scatter area vs forward scatter peak linear and gated on the single-cell population (Figure 4A). The quality of the sort was confirmed by microscopic analysis. To increase organoid-forming efficiency and to avoid anoikis, we used nicotinamide and Rho kinase inhibitor for this experiment.

Sorted Selleck MS 275 cells (0.1%) formed organoids ( Figure 4B) that could be expanded at a 1:5 ratio on a biweekly basis ( Figure 4C). They expressed the gastric markers MUC5AC, PGC, SST, MUC6, TFF1, and TFF2, but not intestinal markers (MUC2, CDX1, and CDX2) as shown by PCR ( Figure 4D). The cellular composition of single-cell–derived organoids was very similar to the one of gland-derived organoids as shown by immunohistochemistry. In the presence of Wnt and nicotinamide, organoids contain PGC-positive chief cells, MUC6-positive mucous neck cells, and very rare SST-positive enteroendocrine cells (gland-type organoids). After 4 days of Wnt withdrawal, MUC5AC-positive mucous pit cells appear (pit-type organoids) ( Figure 4E). Quantifications corroborated these results ( Supplementary Figure 3). In summary, we did not observe any differences between single-cell– and gland-derived organoids in terms of longevity, expansion rate, marker gene expression, or composition Ipilimumab in vivo of cell types. Cultures shown in Figure 4E

are 7 months old, showing that the different cell lineages are maintained over time. We conclude that the single cells behaved as multipotent stem cells. Treatment of gastric cancer patients depends on the availability of tests for drug discovery and sensitivity. Currently, gastric cancer cell lines are available, but no system allows comparison of cancerous and normal cells from Carbohydrate the same patient. Having established the culture condition

for human normal organoids, we reasoned that human gastric tumors also could be expanded under the same conditions. To establish the culture, cells were isolated from the tumor using collagenase and hyaluronidase, seeded into Matrigel, and embedded in ENRWFG_A medium. In parallel, organoids were established from normal tissue (Figure 5A). Chromosomal metaphase spreads were obtained from the tumor organoids ( Figure 5B). Seven spreads were counted and showed aneuploidy with chromosome numbers between 70 and 160. Tumor cultures were reminiscent of the original tissue in terms of morphology shown by H&E staining and p53 accumulation as shown by p53 staining ( Figure 5C, upper panel). To further analyze the possible mutation of the p53 pathway in this tumor, we used nutlin-3, which inhibits the interaction between p53 and MDM2 and thereby induces cell-cycle arrest. Nutlin-3 requires functional p53 and MDM2 for its activity, thus cancer cells with mutated p53 are not affected by this compound. 20 As expected, the normal organoids were strongly inhibited in their growth by nutlin-3, as quantified by a luciferase-based assay.