CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells

– Colorectal cancer, or CRC cells, travel through the circulatory system and metastasize into other organs, such as the liver, lungs, bones, and brain. To study cancer progression, we use an organoid, a 3D in-vitro model that can mimic the organotypic nature of colorectal cancer. Begin by extracting CRC tumors from an anesthetized mouse.

Mince the tumor into small pieces. Now, add the tissue pieces to a tube containing an organoid culture medium including collagenase. Collagenase breaks the tumor clumps, resulting in single-cell suspension of tumor cells.

Next, seed the cell suspension onto an extracellular medium, or ECM-coated plate, and incubate overnight. Add more cell suspension with ECM-containing medium in the wells. Upon embedment into an ECM layer, cancer cells interact with the components of the matrix. These interactions help the CRC cells grow with high efficiencies and self-organize to form organoids.

Finally, collect the organoids using a cell scraper and store them in a microfuge tube for further analysis. In the following protocol, we prepare 3D organoids from colorectal cancer to be used as an in-vitro model to study cancer progression and metastasis.

– To generate human colorectal cancer cell organoids, first add 150 microliters of artificial extracellular matrix per well to a 12-well plate on ice. When all of the wells have been coated, incubate the plate for 30 to 60 minutes in a 37 degrees Celsius and 5% CO₂ incubator to solidify the gels.

Next, re-suspend the patient-derived tumor xenograft cell pellet in fresh colorectal cancer organoid culture medium supplemented with 5% fetal calf serum, or FCS, to achieve a 3 times 10 to the 5 cells per milliliter concentration and seed 1 milliliter of cells onto each artificial extracellular matrix. Then, return the cells to the cell culture incubator.

The next morning, carefully transfer the culture supernatants, including the floating cells, into 1.5 milliliter microcentrifuge tubes for their centrifugation and re-suspend the pellets in 70 microliters of fresh artificial extracellular matrix on ice. To increase colorectal cancer cell viability, overlay the floating tumor cell containing artificial extracellular matrix back onto the artificial extracellular matrix coated wells and incubate the tumor cell cultures for 30 minutes in the cell culture incubator. When the new extracellular matrix gel has solidified, feed the cultures with 1 milliliter of fresh colorectal cancer organoids cell culture medium supplemented with 1% FCS and return the cells to the cell culture incubator.