DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Harvest organoids after 4–7 days of growth

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Harvest organoids after 4–7 days of growth

Publication protocol

Generating Human Fundic Gastric Organoids: Human fundus was collected from sleeve gastrectomies (IRB protocol number: 2015-5537, University of Cincinnati and 2014-0427, Cincinnati Children's Hospital Medical Center), and gastric glands were generated as previously described.28, 29 Briefly, epithelial tissue was separated from the muscle layer, cut into small fragments, and washed in Dulbecco phosphate-buffered saline (DPBS) without Ca2+/Mg2+. Tissue fragments were placed in a buffer containing collagenase (1 mg/mL) from Clostridium histolyticum and bovine serum albumin (2 mg/mL) for 30 minutes at 37°C. Gastric glands were suspended in 50 μL Matrigel (Thermo Fisher Scientific, Waltham, MA) and cultured in freshly generated human gastric organoid media (DMEM/F12 (Thermo Fisher Scientific), HEPES (10 mmol/L), 1X L-glutamine (Thermo Fisher Scientific), 1X Pen/Strep, 1X N2 (Thermo Fisher Scientific), 1X B27 (Thermo Fisher Scientific), N-acetylcysteine (1 mmol/L; Sigma-Aldrich, St Louis, MO), nicotinamide (10 mmol/L; Sigma-Aldrich), epidermal growth factor (50 ng/mL; PeproTech, Rocky Hill, NJ), noggin (100 ng/mL; PeproTech), R-spondin conditioned media, wnt conditioned media, FGF10 (200 ng/mL; PeproTech), gastrin (1 nmol/L; Tocris Bioscience, Bristol, United Kingdom), Y-27632 (10 μmol/L; Sigma-Aldrich), 1X amphotericin B/gentamicin, 1X kanamycin). Organoids were harvested after 4–7 days of growth.

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Manufacturer protocol

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