Metastatic Cancer Cell Colony Isolation from Chicken CAM: A Procedure to Isolate Cancer Cells from Shell-less Egg Cultures
Transcript
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.
