Gibco™Advanced DMEM/F-12

3D Cell Culture Media Human pancreatic organoids

Experiment
3D Cell Culture Media Human pancreatic organoids
Product
Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

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.


Full paper   Login or join for free to view the full paper.

Reviews

Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media Human pancreatic organoids using Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™Advanced DMEM/F-12 below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Human pancreatic organoids using Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms