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

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.