The chicken chorioallantoic membrane (CAM) serves as a vital structure for gas and nutrient exchange in developing embryos. This article outlines a method for creating a CAM cancer model in vitro, detailing the injection of cancer cells into the CAM to study tumor formation and metastasis.
The chicken chorioallantoic membrane, or CAM, of a fertilized chicken egg is the extra-embryonic membrane supported by a rich vascular network. This highly vascularized membrane lining the non-vascular shell membrane facilitates the exchange of gases and nutrients with the developing embryo.
To generate a CAM cancer model in vitro, begin with a viable, fertilized, shell-less chicken embryo bearing a fully developed CAM in a dish.
Next, assemble the injection apparatus containing a tubing attached to a syringe carrying a single-cell suspension of transduced cancer cells in a suitable chilled buffer. Connect a customized glass needle to the tubing for high precision delivery during subsequent injection.
Now, transfer the embryo under a microscope. Identify a suitable vein close to the bifurcation point on the CAM surface. Inject the cancer cell suspension into the selected vein and ensure complete delivery. Gently retract the needle. Cover the embryo and incubate it under appropriate conditions.
The dense vasculature within the mesodermal layer harbors the injected tumor cells, facilitating primary tumor formation. Eventually, some of these cells migrate through the vasculature and extravasate, invading the surrounding stroma, where they proliferate to form metastatic foci.
For IV injection, concentrate cells to 0.5 to 1 million cells per milliliter using ice-cold 1x PBS to dilute or resuspend the cells. Expect that approximately 1 milliliter of cell suspension will be needed for every 10 embryos. To assemble the injection apparatus, mount a needle onto the syringe and then extend the syringe needle with a 3 to 5-centimeter long piece of tubing.
Break the tip of the borosilicate needle using fine forceps. Load the syringe with 50 to 200 microliters of the cancer cell suspension and insert the borosilicate needle into the tubing. Inspect the needle for cell clogging and air bubbles. If cell clogging is observed within the capillary, remove the capillary, resuspend the cells, and replace it with a new capillary.
Use the plunger to push out any bubbles. Remove the cover lid and transfer the embryo under the stereoscope. Identify the appropriate vein to be injected on the CAM surface, which is normally only slightly wider than the diameter of the borosilicate needle tip and located midway between the embryo and the weighing dish wall.
It is generally easier to inject at the point immediately adjacent to the vein bifurcation. Press the tip of the needle against the blood vessel wall and apply gentle pressure in the same direction as the blood flow. If necessary, use a cotton swab to help anchor or stabilize the vessel that is being injected. Gently depress the syringe plunger for 2 to 10 seconds until the desired volume is injected.
Discard the embryo if excessive bleeding or clear liquid accumulation appears at the injection site. Remove the needle from the CAM and gently dab the injection site with a cotton swab to remove any blood or excess cancer cells. Cover the injected embryo in the weighing dish with the lid and return it to the incubator. Repeat the procedure until all embryos are injected.
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