Biozellen™ Colorectal Cancer Organoid Medium
Biozellen™ Colorectal Cancer Organoid Medium is a chemically defined cell culture medium for the establishment and maintenance of human Colorectal Cancer organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.
Storage & Stability
Cancer Organoid Basal Medium A
4℃， 3 months or
Protocol for Establishment of Patient-Derived Colorectal Cancer Organoids
1. Collect primary human Colorectal cancer tissue pieces in ice-cold Primary Tissue Storage Solution with conical tubes. Keep tissue samples at 4°C until the start of the isolation.
2. Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.
3. Rinse the tissue with Organoid Basal Solution or DPBS twice.
4. Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.
5. Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.
6. Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.
7. Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.
8. Aspirate the supernatant and resuspend the pellet in Organoid Basal Solution or DPBS, centrifuge at 250g for 3min at 4°C, repeat this step once more time.
9. Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.
CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.
10. Plate the Matrigel containing organoids at the bottom of 24-well or 48-well cell culture plates in droplets of ~30 μL each around the center of the well..
CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.
11. Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.
12. Prepare the required amount of Colorectal cancer organoid complete medium.
13. Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of Biozellen™ Colorectal Cancer Organoid Medium to each well.
CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.
14. Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.
15. Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.
16. Closely monitor the organoid formation. Ideally, patient-derived Colorectal cancer organoids should be passaged for the first time between 7 and 10 d after the initial plating.
17. Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.
18. Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.
19. Centrifuge organoids at 250g for 3 min at room temperature.
20. Aspirate the supernatant and split organoids using either Organoid Digestion Solution or by mechanical disruption. For Organoid Digestion Solution -based cell dissociation, resuspend the pellet in 0.2 mLof Organoid Digestion Solution , pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥3 times every 1 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Organoid Basal Solution . Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.
CRITICAL: Do not dissociate in Organoid Basal Solution for >3 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.
21. After shearing is complete, wash once by topping up with 1 mL of Organoid Basal Solution B
or DPBS, and centrifuge at 250g for 3 min at room temperature.
22. Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.
23. Pre-warm Colorectal Cancer Organoid Medium at 37 °C.
24. After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.
25. Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.
For Research Use Only