Colorectal Cancer Organoid Generation

Patient-derived colorectal cancer organoids were generated and cultured as described earlier [21 (link), 22 (link)]. In brief, the tissue was cut into small pieces and dissociated at 37 °C. Dissociated cells were passed through a 30 and 100 μm cell strainer and collected in advanced DMEM/F12 medium (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US) supplemented with Glutamax (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US), penicillin/streptomycin, HEPES, N-acetylcysteine (Merck Chemicals GmbH, Darmstadt, Germany), N-2 supplement (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US), B-27 supplement (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US), EGF (PeproTech GmbH, Hamburg, Germany), Y27632 (Absource Diagnostics GmbH, Munich, Germany), and amphotericin (Sigma-Aldrich Chemical, St. Louis, Missouri, US) and embedded in matrigel (Corning B.V., Amsterdam, Netherlands). For subcultivation, organoids were removed from matrigel and dissociated into small organoids using TrypLE (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US) and then transferred into fresh matrigel. For coculture experiments, organoids were dissociated into small organoids and 2,500–5,000 small organoids were seeded onto the peritoneum.

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Mönch D., Koch J., Maaß A., Janssen N., Mürdter T., Renner P., Fallier-Becker P., Solaß W., Schwab M., Dahlke M.H., Schlitt H.J, & Leibold T. (2021). A human ex vivo coculture model to investigate peritoneal metastasis and innovative treatment options. Pleura and Peritoneum, 6(3), 121-129.

Publication 2021

advanced DMEM/F12 medium Glutamax penicillin/streptomycin HEPES N-acetylcysteine N-2 supplement B-27 supplement amphotericin matrigel TrypLE

Amphotericin Cell Coculture Colorectal cancer Diagnostics Hepes Matrigel N acetylcysteine Organoids Patient Penicillin Peritoneum Streptomycin Supplement Tissue Y27632

Corresponding Organization : Robert Bosch Hospital

Other organizations : Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tübingen

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Variable analysis

independent variables

Tissue dissociation conditions (temperature, duration)
Cell strainer size (30 μm, 100 μm)
Medium composition (advanced DMEM/F12, Glutamax, penicillin/streptomycin, HEPES, N-acetylcysteine, N-2 supplement, B-27 supplement, EGF, Y27632, amphotericin)
Matrigel embedding
Organoid dissociation and subculturing (TrypLE treatment)

dependent variables

Organoid growth and development

control variables

Cell culture conditions (temperature, CO2 levels, humidity, etc.)
Dissociation and culture media components
Matrigel embedding protocol

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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