Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Use of pre-warmed 24-well plate Add fresh medium was every 2-3 days.	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
Use of pre-warmed 24-well plate Add fresh medium was every 2-3 days.
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: Normal gastric organoids were derived from body (BO) and antral (AO) type mucosa and fresh tissues were processed using a previously published protocol (Barker et al., 2010, Bartfeld et al., 2015). Briefly, normal tissues were washed with PBS containing 1x Penicillin/ Streptomycin (P/S) twice, followed by removing the muscle layer and mucus using scissors. Normal tissues were then cut into 1-3 mm2 pieces and incubated in PBS buffer containing 10mM EDTA, 0.5mM DL-dithiothreitol, 1x P/S and 1μg/ul Primocin at 4°C for 45min. Tissue-pieces were then carefully transferred to a sterile 10cm dish and gastric glands were released by pressing the tissues using a glass-slide. Relased gastric glands were washed twice with PBS, followed by centrifugation at 200 g for 5min and resuspened with Matrigel. A drop of 50μl matrigel-cell mixture was added to each well of a pre-warmed 24-well plate (∼8-16 drops per sample). After the drops solidified, 500μl of standard gastric organoid medium (advanced DMEM/F12, 1x GlutaMax, 1x HEPES, 1x P/S, 50% Wnt3a, 10% RSPO-1, 10% Noggin, 1xB27, 50ng/mL EGF, 200ng/mL FGF10, 1mM N-Acetylcystenine, 1nM Gastrin, 2μM A83-01), 10μM Y-27632 and 1μg/ul primocin were added to each well. Fresh medium was added every 2-3 days and cystic normal orgnaoids appeared after 5-7 days. 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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