Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

The chicken chorioallantoic membrane, or CAM, is a highly vascularized membrane found in the eggs of certain vertebrates like chickens. This extra-embryonic membrane lines a non-vascular shell membrane and functions as the site of exchange of gaseous oxygen and carbon dioxide between the embryo and the environment.

To generate a CAM cancer model, begin with a viable, fertilized chicken egg bearing a fully developed CAM with a partially opened shell that exposes its branching vasculature. Confirm that the CAM is detached from the shell membrane.

Place a sterile non-stick ring over a blood vessel branch point. Using a glass rod, gently rub the membrane inside the ring to scrape its outer epithelial layer and expose the underlying mesodermal layer carrying the vasculature. Pipet a chilled suspension of cancer cells mixed with a suitable extracellular matrix into the ring. The ring restricts cellular dispersal.

Allow the matrix to solidify to secure the cells within. Seal the eggshell opening and incubate the egg under appropriate conditions. The dense vasculature within the mesodermal layer supplies oxygen and essential nutrients to the cancer cells, thus providing a suitable microenvironment for their growth and expansion.

To prepare a cancer cell suspension for transplantation, harvest the cells from the cancer cell culture of interest and sediment the cells by centrifugation. Aspirate the supernatant and mechanically resuspend the cells in the residual medium. Place the cell suspension on ice and use a pipette to measure the volume of cells in medium. Then, resuspend the cells at a concentration of 1 to 2 million cells in 20 to 100 microliters of medium, supplemented with 2.7 to 4 milligrams per milliliter extracellular matrix and any growth factors or other additives of interest.

For cancer cell implantation, using a non-stick ring, place up to 6 eggs to be implanted onto the egg rack and roll the edges of the transparent film dressing toward the open window to remove the film from the shells. Check whether the eggs are viable and healthy. Ideal eggs will have a large vessel with smaller branching vessels within the center of the opened area.

Using curved iris forceps, place a sterile non-stick ring onto the CAM over the vessel, ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM and pipet the cancer cell suspension into the center of the ring. When all of the cells have been delivered, seal the opening with a one quarter piece of 6 by 7 centimeter transparent film dressing and label the egg with an appropriate implant designation. Then, place the egg securely in the incubator, with the opening of the shell facing upright.