The chicken chorioallantoic membrane (CAM) is a tri-layered structure that provides a conducive environment for tumor cell colonization. This article details a method for isolating cancer cells from the CAM, utilizing collagenase treatment and selective growth media.
The chicken chorioallantoic membrane or CAM is a tri-layered membrane located just underneath the eggshell. It consists of an outer ectoderm, a highly vascular mesoderm, and the innermost endoderm.
The CAM mesoderm comprises of blood vessels, fibroblasts, and stromal components, and thus provides a favorable environment for tumor cell colonization.
To isolate colonized cancer cells from CAM, begin by taking a shell-less egg culture pre-injected with fluorescently labeled cancer cells in a dish. Visualize the egg culture under a fluorescence microscope. Locate the fluorescent and compact metastatic cancer colonies.
Gently excise the CAM tissue containing metastatic colonies of interest. Transfer the excised CAM tissue into a microcentrifuge tube. Mince the tissue into small pieces to make the CAM accessible to enzymatic treatment in the subsequent steps.
Supplement the tube with collagenase enzyme and incubate. Collagenase degrades the collagen protein in CAM mesoderm and initiates cancer colony dissociation. Centrifuge the suspension to pelletize the cancer cells, remnants of CAM tissue, and CAM fibroblasts. Discard the collagenase-containing supernatant.
Add a selective growth media and transfer the suspension into a fresh culture plate. This media promotes the growth of suspended cancer cells over other cells. Aspirate the cancer cells and store them for further downstream analysis.
At day 5 post injection, remove the embryos from the incubator and inspect and locate the embryo CAMs for metastatic colony distribution. Under the dissection microscope, gently pull the CAM tissue that contains the metastatic colony of interest upwards using fine forceps and cut it off with surgical scissors.
Transfer the CAM tissue into an empty sterile 1.5-milliliter tube and close the tube lid. Repeat the excision procedure until all the colonies of interest are collected into separate tubes. Gently mince the CAM tissue in a microcentrifuge tube using a separate sterile 18G needle for each colony. Add 100 microliters of 1x collagenase solution and incubate for 30 minutes at 37 degrees Celsius.
Spin down the cells and CAM tissue at 300 x g for 5 minutes at ambient temperature. Aspirate the collagenase solution and resuspend the cells in complete media for the cell line of interest. Then, spin the cells and tissue again.
Resuspend cells and tissue pieces in 1 milliliter of complete media and selection factor if any. Then, transfer into a single 12-well tissue culture dish well. For the next one to three weeks, monitor the cancer cells daily for growth and contamination. When the cells reach 70% to 80% confluency, transfer them into a larger volume culture dish. Proceed to sequencing or the next round of selection as soon as adequate cancer cell numbers are reached.
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