Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Cultrex growth factor reduced BME type 2 in advance, on ice or in fridge	After seeding, inverted and maintained plates at 37 °C to let the BME solidify

Upstream tips
Defrost the Cultrex growth factor reduced BME type 2 in advance, on ice or in fridge
Protocol tips
After seeding, inverted and maintained plates at 37 °C to let the BME solidify

Publication protocol

Tissue Processing: Patient material was collected in Advanced DMEM/F12 (Life Technologies; 12634-034), supplemented with 1× GlutaMAX (adDMEM/F12; Life Technologies; 12634-034), penicillin-streptomycin (Life Technologies; 15140-122) and 10 mM Hepes (Life Technologies; 15630-056) (designated +/+/+ hereinafter). For collection of patient material, 100 µg/mL Primocin (InvivoGen; ant-pm1) was added. Material was cut into small fragments. Random pieces of ∼5 mm3 were stored at −20 °C for DNA isolation or fixed in formalin for histopathological analysis. Fragments were incubated at 37 °C in 1 mg/mL collagenase (Sigma-Aldrich; C9407). Digested tissue was sheared using 5-mL pipettes. Cell suspension was diluted with 10 mL of +/+/+ and strained over a 100-µm EasyStrainer filter (Greiner; 542000) and centrifuged at 300 × g. The resulting pellet was resuspended in ice-cold 70% 10 mg·mL−1 cold Cultrex growth factor reduced BME type 2 (Trevigen; 3533-010-02) in organoid medium. Droplets of ∼10 µL were plated in preheated suspension culture plates (Greiner; M9312). Plates were inverted and maintained at 37 °C to let the BME solidify. After 30 min, prewarmed organoid medium was added to the plates. For the first week, 10 µM rho-associated kinase (ROCK) inhibitor (Y-27632; Abmole Bioscience; M1817) was added to the medium.
Organoid Culture: Organoids were grown in +/+/+ supplemented with different subsets of growth factors, depending on whether wild-type or tumor organoids were established. For organoids derived from wild-type tissue, medium consisted of Wnt3a-conditioned medium (50% vol/vol), plus +/+/+ containing 1× B27 supplement (Life Technologies; 17504-044), 1,25 mM N-acetyl-l-cysteine (Sigma-Aldrich; A9165), 10 mM nicotinamide (Sigma-Aldrich; N0636), 50 ng/mL human EGF (PeproTech; AF-100-15), 500 nM A83-01, 100 ng/mL human FGF10 (PeproTech; 100-26), 1 µM prostaglandin E2 (Tocris Bioscience; 2296), 10 nM gastrin (R&D Systems; 3006), 4% (vol/vol) RSPO, and Noggin (produced via the r-PEX protein expression platform at U-Protein Express BV). This medium was termed complete medium (CM). Tumor organoids were grown in parallel in 2 types of tumor organoid media, tumor medium 1 (TM1) and tumor medium 2 (TM2). TM1 was identical to CM, the only difference being the absence of EGF and PGE2. The difference between TM2 and CM was the absence of PGE2, A83-01, and Wnt3a-conditioned medium. For passaging, organoids were collected, washed, and disrupted either by mechanical shearing or digestion with TrypLE Express (Life Technologies; 12605-010). After passaging, organoid fragments were replated in fresh BME, and 10 µM Y-27632 was added to prevent cell death. The organoid passaging procedure is described in detail in SI Appendix.


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