OncoPro™ Tumoroid Culture Medium Kit - FAQs

OncoPro Tumoroid Culture Medium Kit has been developed and optimized for the expansion and maintenance of existing tumoroid cultures. It has been used to successfully derive new tumoroid lines from patient tumor resections, dissociated tumor cells, and patient-derived xenografts, however the success rate of new derivations is highly variable and dependent on the quality of the starting tissue, tissue type, donor, etc.

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The complete OncoPro Tumoroid Culture Medium may appear yellow when it is first prepared. This is normal and does not indicate any issues with buffering or pH. The yellow color will dissipate somewhat after refrigeration and/or incubation.

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Yes, the complete OncoPro Tumoroid Culture Medium should be supplemented with freshly thawed 10 µM Y-27632 for every feed.

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We recommend using StemPro Accutase Cell Dissociation Reagent (Cat. No. A1110501) for passaging tumoroids.

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Tumoroid cultures should be passaged when the average tumoroid size is 200-300 µm in diameter. This is typically every 7 days, but depending on the tumoroid line, may occur anywhere between 4-14 days after seeding. We do not recommend passaging when the average tumoroid size is <100 µm or >400 µm in diameter.

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We do not recommend passaging tumoroids before they reach at least 200 µm in diameter. Over-dissociation can negatively impact the viability and subsequent expansion of tumoroid cultures.

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For colorectal cancer tumoroid lines, we recommend a split ratio of 1:7. We recommend a seeding density of 0.125 x 10E6 cells/mL to 0.2 x 10E6 cells/mL for suspension culture. We recommend a seeding density of 5 x 10E4 cells/dome for embedded cultures for 50µL domes.

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Suspension conditions eliminate the need for manually embedding cells in basement membrane extract domes. As a result, suspension cultures are less labor intensive, more cost efficient, more scalable, and more compatible with automation than embedded cultures.

We have not found any significant differences in morphology, growth rate, mutational profile, or gene expression signature between tumoroids cultured in parallel, in suspension and embedded culture conditions.

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We routinely freeze tumoroids at 1-2 x 10E6 cells/mL in Recovery Cell Culture Freezing Medium. Please see the user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0029372-OncoPro-Tumoroid-Culture-Medium-UG.pdf) for guidance on recovering cryopreserved tumoroids.

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We have data demonstrating that morphology, growth rate, mutational profile, and gene expression profile are conserved in tumoroid lines cultured for over 250 days (35+ passages post-thaw from bank).

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For suspension culture, we recommend the use of Nunc Non-Treated Multidishes and Nunc Non-treated Flasks, specifically Nunc Non-Treated 6-well plate (Cat. No. 150239), Nunc Non-treated T25 EasyFlask, Filter Cap (Cat. No. 169900), and Nunc Non-treated T75 EasyFlask, Filter Cap (Cat. No. 156800). For embedded culture, we recommend the use of Nunc Cell-Culture Treated Multidishes, specifically Nunc Cell-Culture Treated 6-well plate (Cat. No. 140675).

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OncoPro Tumoroid Culture Medium Kit has been developed and optimized for the culture of primary patient-derived tumoroids. It has not been validated for the culture of healthy primary tissues or cells.

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OncoPro Tumoroid Culture Medium does not contain any activators of the Wnt signaling pathway.

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OncoPro Tumoroid Culture Medium Kit is a novel media system that was specifically developed for the expansion of patient-derived tumoroids. The exact formulation of OncoPro Tumoroid Culture Medium is proprietary, however unlike other published methods, OncoPro Tumoroid Culture Medium does not rely on the addition of Wnt, R-spondin, or Noggin for long term maintenance of patient-derived tumoroids. OncoPro Tumoroid Culture Medium also does not contain any small molecule inhibitors.

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Yes, OncoPro Tumoroid Culture Medium Kit has been validated for the culture of colorectal, lung, pancreatic, and head and neck tumoroids. Some cancer indications may require supplementation with additional growth factors.

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The requirements for additional growth factors with OncoPro Tumoroid Culture Medium depend upon the type of cancer. Colorectal tumoroids do not require supplementation with any additional growth factors. Lung and head and neck tumoroids require the addition of heat stable FGF10. Pancreatic tumoroids require the addition of heat stable FGF10 and gastrin. The requirements for other cancer indications will need to be determined empirically.

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Yes, we recommend preparing aliquots of the OncoPro Supplement and B-27 Supplement and storing at -20 degrees C. Please see the user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0029372-OncoPro-Tumoroid-Culture-Medium-UG.pdf) for guidance on aliquoting and freezing. Avoid additional freeze-thaw cycles. We do not recommend preparing and freezing complete OncoPro Tumoroid Culture Medium.

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The shelf life of the complete OncoPro Tumoroid Culture Medium is 1 week at 4 degrees C.

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Some tumoroid lines can be immediately transitioned into OncoPro Tumoroid Culture Medium suspension culture. Some more sensitive lines may benefit from a more gradual, stepwise transition. You can find additional guidance in the OncoPro Tumoroid Culture Medium Kit user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0029372-OncoPro-Tumoroid-Culture-Medium-UG.pdf).

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Yes, we have successfully transitioned tumoroids that were previously cultured in a number of different media systems into OncoPro Tumoroid Culture Medium.

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We recommend feeding cells every 2-3 days until tumoroids reach an average size of 200-300 µm, at which point they are ready to be passaged or harvested for downstream assays.

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We recommend using heat stable FGF10 for cancer indications requiring supplementation with FGF10. This allows for the use of lower concentrations of growth factor and is more cost effective than using wild type FGF10. If wild type FGF10 is used, concentrations should be increased to 100 ng/mL throughout the protocol. We have successfully cultured tumoroids in medium containing 100 ng/mL wild type FGF10 without causing significant differences in morphology, growth rate, mutational profile, or gene expression signature compared to culturing in medium with 10 ng/mL heat stable FGF10.

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We have successfully used various basement membrane extracts (BMEs) for culturing tumoroids in suspension. BME used in these workflows must have a concentration >10 mg/mL prior to dilution. Some ready-to-use formulations may not be suitable for these applications due to their low protein concentration.

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Yes, the protocol calls for the addition of 2% v/v diluted basement membrane extract (BME) to suspension cultures. This promotes the formation and stability of 3D tumoroids. Tumoroids are still free-floating at this concentration as it is much lower than the concentration used for embedded cultures.

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We have data demonstrating that morphology, growth rate, mutational profile, and gene expression profile are conserved between cells grown in embedded and suspension culture conditions.

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Yes, we have data showing that some tumoroids cultured using OncoPro Tumoroid Culture Medium Kit are tumorigenic and can generate tumors in mouse xenograft models. Xenograft success rates will depend on the characteristics of the tumoroid model and the mouse strain used.

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Yes, we have supporting protocols available for transitioning suspension tumoroid cultures to traditional 2D assays such as migration-invasion assays.

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