Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Enrich for tumor organoids portion by manual selection under the microscope during the first 2-3 weeks of culture	Collect organoids to fix them in 4% paraformaldehyde for 1hr, embed in 2% agarose gel, or directly fix in the matrigel in formalin for generation of paraffin blocks, sectioning and staining

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Enrich for tumor organoids portion by manual selection under the microscope during the first 2-3 weeks of culture
Downstream tips
Collect organoids to fix them in 4% paraformaldehyde for 1hr, embed in 2% agarose gel, or directly fix in the matrigel in formalin for generation of paraffin blocks, sectioning and staining

Publication protocol

Establishment of organoid cultures: The establishment of tumor organoid cultures was complicated by various factors, including the stromal composition of the sample, normal cell contamination and overgrowth, and the sparse number of cancer cells in some molecular subtypes (e.g., diffuse type). Therefore, we modified the protocol (Barker et al., 2010, Bartfeld et al., 2015) by incorporating variable digestion times depending on the tumor character; avoiding sampling from the mucosal side; closely monitoring the organoids during early culture for normal contamination and manually re-plating only the tumor cells; and adding Nutlin3a for the enrichment of tumor organoids carrying TP53 mutations. The modifications and special conditions used to establish each tumor organoid line is shown in Table S2. Briefly, for tumor organoids, tumor tissues were rinsed with advanced DMEM/F12 containing 1xP/S twice, and then were minced using scissors and blades in a 10cm dish with digestion buffer (advanced DMEM/F12 containing 1x P/S, 1μg/μl primocin, 2.5% FBS, 0.6mg/mL collagenses, 20μg/mL hyaluronidase and 10μM Y-27632). Tumor cells were released from bulk tissues using digestion buffer at 37°C, with variable incubation times ranging from 1 hour to overnight, depending on the stiffness of the tissue. Tissue debris was removed by passing the mixture through a 100 μm cell strainer. Cell clusters were washed twice using AdDMEM medium by centrifugation at 1000 g for 5min, and resuspended in Matrigel, and plated in a 24-well plate as normal glands. Primocin was added during the first 2 weeks of all organoid cultures. For tumor organoids with normal organoid contamination, we enriched for the tumor organoids portion by manual selection under the microscope during the first 2-3 weeks of culture. Briefly, cell mixtures were released from the matrigel using a cell recovery solution (BD bioscience, 500 μl/drop) at 4°C for 20 min. The cell suspension was transferred to a pre-wetted 10cm culture dish using a wide-bore tip to avoid breaking of cystic normal organoids. Using a pre-wetted 200μl tip, under a microscope, as many tumor clusters as possible were manually picked while avoiding the cystic organoids. The picked tumor clusters were embedded in matrigel as before. This process of manual picking of tumor organoids was repeated as necessary if normal organoids remained. TP53 immunostaining was performed on tumor tissues in parallel with cell isolation. If the immunostaining was suggestive of TP53 mutation by either stabilization/strong overexpression or complete absence of nuclear protein staining, then 10μM nutlin3a was added to the tumor organoid cultures. Selection with nutlin3a was maintained during the cultures unless specified.
Passage of the organoid cultures was performed two weeks after isolation. Normal organoids embedded in matrigel were disrupted mechanically by pipetting using a pre-wetted fire-polished glass Pasteur pipette. Tumor organoids were passaged by incubating in trypsin at 37°C for 3-5 min. The cell mixture was washed and embedded in matrigel at a 1:2 ratio. The culture medium was changed every 2-3 days and 10μM Y-27632 was added after passage. To cryo-preserve organoids for long-term storage, organoids were stocked in 1.5ml cryotubes with culture medium containing 5% DMSO. For DNA, RNA and protein extraction from organoids, organoids were released from the matrigel using a cell recovery solution at 4°C for 30 min, followed by washing with PBS twice before adding lysis buffer. Niche factors, including Wnt3a, RSPO-1 and Noggin were prepared as conditioned medium for organoid cultures. In brief, L-Wnt3a cell line was cultured in DMEM culture medium containing 10% FBS, P/S and zeocin. Cells were plated in 150mm2 culture plates and left in a 37°C incubator for 7 days before collecting the supernatant as conditioned medium. 293T/17 R-Spondin-1 cell line was cultured in Advanced DMEM/F12 medium containing 10% FBS, P/S and zeocin for making conditioned medium. A pcDNA3.1-based mammalian expression vector containing mouse Noggin cDNA was transiently transfected in to 293T/17 cell line for production of Noggin conditioned medium. After transfection, the cells were cultured in Advanced DMEM/F12 medium for 7 days. All conditioned media was collected by centrifugation at 1500rpm for 5min and filtered using a 0.2μm filter-top to remove cell debris. Long-term culture of tumor organoids was performed in 6 cases, in which organoids were continuously passaged for over 6 months before collection of samples for histological and genomic analysis. Fibroblasts were cultured from patients’ cancer or gastric mucosal tissues by plating the residual stroma tissues on tissue culture plates supplemented with F12/DMEM medium containing 10% FBS, P/S, 50ng/mL EGF, 10μg/mL insulin and 5μg/mL transferrin. All cultures were routinely tested every two months to ensure they were mycoplasma free. For histological examination of the organoids, the organoids were either released from the matrigel using cell recovery solution (BD bioscience) and fixed in 4% paraformaldehyde for 1 hour, embedded in 2% agarose gel, or directly fixed in the matrigel in formalin for generation of paraffin blocks, sectioning and staining.


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