Chick Chorioallantoic Membrane or CAM Tumor Model: An In Ovo Method to Simulate Perineural Invasion of Tumor Cells

Overview

In this video, we describe the method to co-culture dorsal root ganglia and cancer cells on chick chorioallantoic membrane or CAM to study perineural invasion.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Grafting DRG on the CAM (estimated timing: 40 min, day 8)

1. Prepare a 6 cm culture dish with HBSS medium to wash the DRGs before implantation. Bring the prepared eggs to the cell culture laminar flow cabinet. Remove the paraffin membrane from the egg (Figure 1A).
2. With fine sterile forceps, gently grasp one DRG from inside the culture medium. Dip it into the HBSS medium to remove the excess medium that contains the fluorescent dye. Hold the DRG very gently; otherwise, it will stick to the forceps. 
3. Place the DRG on the CAM, being careful not to puncture the membrane (Figure 1B-C). If necessary, use another pair of forceps to help detach the DRG from the tip of the forceps when placing it on the CAM. 
   NOTE: Keeping the DRG wet with HBSS medium will also facilitate detachment from the forceps.
4. Cover the egg with a sterile transparent film dressing. Cover all the windows and punctures made in the eggshell to avoid bacterial contamination (Figure 1D).
5. After grafting DRGs in all eggs, incubate the eggs in a humidifying incubator at 38 °C and 54% humidity for 2 days, without rotation.

2. Grafting Tumor Cells on the CAM (estimated timing: 1 h 30 min, day 10)

1. At 48 h before grafting the cells, plate the cells needed in culture plates. Calculate 0.5 to 1 x 10⁶ cells per egg for UM-SCC-1 cells in order to generate three-dimensional tumors in the CAM. Be aware that cell number may vary by cell line.
2. Aspirate medium and add DMEM culture medium supplemented with 1% Pen/Strep plus 10% FBS and 2.5 µg/mL of green fluorescent dye. Incubate for 1 h at 37 °C in the cell culture incubator. Then, check fluorescence intensity on the microscope, aspirate medium, wash once with 1X PBS, and add 0.25% trypsin for up to 10 min. Neutralize trypsin with DMEM supplemented medium. 
3. Centrifuge at 250 x g for 4 min to form a cell pellet. Aspirate DMEM medium and resuspend in HBSS to wash the excess fluorescent dye. Count cells using a hemocytometer. 
4. Bring the eggs to the cell culture laminar flow cabinet. With scissors and forceps, open the transparent film dressing that covers the egg (Figure 1E-F).
5. Centrifuge the calculated number of cells again, aspirate the HBSS medium, and resuspend at a final concentration of 0.5 to 1 x 10⁶ cells per 5 µL of the same medium (Figure 1G). Prepare the amount needed for the total number of eggs (5 µL of cell suspension per egg).
6. Place 5 µL of cell solution onto the CAM, about 2 mm from the DRG (Figure 1H). Keep uniform distances between the DRG and cells. Be very careful not to disturb the egg to minimize spreading of the cells. 
   NOTE: To start cell implantation, choose eggs on which the CAM surface is visually dry. If the surface is too wet, cells can spread and do not form regular tumors.
7. Cover the egg with a new film dressing as in step 1.4. Graft the cells in all eggs. Incubate the eggs in a humidifying incubator at 38 °C and 54% humidity for 7 days, without rotation.

Representative Results

Figure 1: Grafting of DRG, cells, and harvesting of CAM: On day 8: A. CAM easily observed after paraffin wax membrane removal. B-C. With fine forceps, DRG is placed onto the CAM. D. Egg is covered with film dressing and put in the incubator; arrows point to the openings that are covered. On day 10: E-F. Film dressing is removed and DRG is located (arrow head on F). G-H. 5 µL of cell solution is dropped onto the CAM at a ~2 mm distance from the DRG. 

Materials

Name	Company	Catalog Number	Comments
Egg incubator	GQF Digital Sportsman	#1502	Egg incubator equipped with automatic rotator digital thermostat temperature and humidity controls
Engraving cutter	DREMEL	#108	Used to drill the egg shell
Extra fine Graefe forceps, curved	Fine Science Tools (FST)	#11151-10	Used to graft DRG on to the CAM on day 8 and to harvest CAMtissue on day 17
Extra fine Graefe forceps, straight	Fine Science Tools (FST)	#11150-10	Used to graft DRG on to the CAM on day 8 and to harvest CAM tissue on day 17
Fertilized Lohmann White Leghorn eggs			Fertilized eggs at early fertilization days preferably on first day postfertilization. Eggs used in this protocol are from Michigan State University Poultry Farm.
Filter Forceps	EMD Millipore	#XX6200006P	Blunt forceps used to remove the egg shell
HBSS (1x)	Gibco	#14025-092	Hank`s Balanced Salt Solution
Paraffin wax membrane	Parafilm laboratory film	#PM-996	Used to temporarily cover the egg openings until DRG grafting on day8
PBS (1x) pH 7.4	Gibco	#10010-023	Phosphate Buffered Saline
Tegaderm Transparent Film Dressing	3M	#9505W	Sterile, 6x7cm used to cover the egg openings during incubation