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Encyclopedia of Experiments

Chick Chorioallantoic Membrane Harvest: A Method to Isolate Tumor Graft-bearing Chorioallantoic Membrane from Chicken Egg

Overview

This video demonstrates the isolation of tumor-bearing chorioallantoic membrane from the chicken embryo. The harvested CAM can be used for further downstream applications.

Protocol

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

1. Harvesting the CAM

  1. Prepare 6-well plates with 4% PFA (paraformaldehyde) pH 7.0; one well per egg, 2 mL per well.
  2. Bring the eggs to a laboratory bench. Using a needle attached to a syringe to perforate the film dressing, drop around 300 µL of PFA over the CAM to slightly stiffen the CAM, thus facilitating the harvesting process. Repeat this for all eggs.
  3. With a scissor, remove the upper half of the eggshell (where the operating window is located) with the CAM attached to it (Figure 1I). Reduce the size of this half to approximately 3 cm in diameter. Grasp the CAM with fine forceps and detach it from the eggshell while placing it into PFA.

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Representative Results

Figure 1
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. On day 17: I-L and M-P demonstrate two different approaches used to harvest the CAM. I. Eggshell is opened with a fine scissor starting on the air sac drilled perforation until the upper half of the egg is removed. J. Eggshell containing the CAM is reduced in size to approximately 3 cm. K-L. With fine forceps, CAM is detached from the eggshell and placed in PFA. M-O. Widening of the operating window is performed to visualize the DRG and cancer cells on the CAM. Arrowhead points to the tumor and arrow points to the DRG. P. The CAM is grasped with fine forceps, cut out with a sharp scissor, and placed in PFA as shown in L

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Materials

Name Company Catalog Number Comments
PFA (paraformaldehyde solution) Sigma-Aldrich # P6148-1KG Dilute in water to make a 4% PFA solution
Fine surgical straight sharp scissor Fine Science Tools (FST) #14060-09 Used to harvest the 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
Extra fine Graefe forceps, straight  Fine Science Tools (FST)  # 11150-10  Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17

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