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CAM Assay
• Chick Chorioallantoic Assay
• Screening assay for potential pro- and anti-angiogenic agents

• Formation of new blood vessels out of pre-existing capillaries
• Assessment is an indirect assessment of the carcinogenicity of a drug
CAM Assay
• Used in the studies of:
• Angiogenic effect of drugs
(by examining the response in the rapidly growing CAM)
• Angiogenesis inhibitors (Fibroblast growth factor-2)
CAM Assay
• Principle:

• Capillaries appear in the yolk sac

of the fertilized egg at 28 hours
and grow rapidly over the next
6-8 days which is the best time
to introduce the test chemical
agent and observe its effect on
the 12th day
Chorioallantoic Membrane
• Develops from 4th to 5th ID in the domestic fowl
(Gallus domesticus)
• Fusion of mesodermal layers of chorion and allantois
• Resembles the primary respiratory organ of the
developing avian embryo
• Serves as an organ for gaseous exchange between
the pores of the eggshell and dense network of
capillaries subjacent in a “placenta-like” fashion.
• It finally covers almost the complete interior surface
of the egg shell by ID 12.
METHODOLOGY: Version 1 (In Ovo)

1. Punch filter paper using a puncher (approx. 5 mm. in diameter).

2. Sterilize by autoclaving or exposing to UV light for at least 15 minutes.
METHODOLOGY: Version 1 (In Ovo)

1. Incubate the fertilized egg for 8-10 days at 37oC and 70% humidity
2. Cut 1x1 cm window in the egg shell to expose the CAM
3. Place the treated filter disc directly onto the CAM
4. Seal the treated eggs with plastic tape and incubate for two days
METHODOLOGY: Version 1 (In Ovo)

5. After 48 hours of incubation, harvest the CAM carefully removing the hard
shell leaving intact the soft membranes covering the embryo
6. Transfer the shell-less embryo to a glass petri dish
7. Examine CAM for the number of blood vessel branch points
METHODOLOGY: Version 1 (In Ovo)

1. Examine the CAM at the site of application for angiogenesis

2. Draw 4 quadrants about the area of the CAM
3. Manually count the blood vessel branch points at each area of the
quadrant counting clockwise
4. CAM is photographed for documentation using 40 to 80 x total
magnification and the ocular lens
METHODOLOGY: Version 1 (In Ovo)
• CAM vascularity can be expressed as % of control

No. of branch points (treated) – No. of branch points (control)

x 100
No. of branch points (control)
Baptista, P., Kanaras, A., Heuer-Jungemann, A., Roma-Rodrigues, C., & Fernandes, A. (2016). Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo. International Journal of Nanomedicine, 2633. doi:10.2147/ijn.s108661
METHODOLOGY: Version 2 (Ex Ovo)
1. Fertilized White Leghorn chicken eggs staged according to Hamburger and
Hamilton are placed in an incubator as soon as embryogenesis starts and
are kept under constant humidity at 37oC.
2. On day 3, a square window is opened in the shell after removal of 2-3 mL
of albumen to detach the CAM from the shell itself and the underlying
CAM vessels are disclosed.
METHODOLOGY: Version 2 (Ex Ovo)
3. The window is sealed with a class and incubation goes on until the day of
the experiment. This technique may preserve amore physiological
environment; however, it limits the are for use and observation.
4. The embryo and its extraembryonic membranes are transferred to a petri
dish on day 3 or 4 incubation and CAM develops at the top as a flat
membrane and reaches the edge of the dish to provide two dimensional
monolayer onto which multiple grafts can be placed.
METHODOLOGY: Version 2 (Ex Ovo)
• Survival rate of eggs cultured ex ovo is the major success limiting step in
this technique
• The ex ovo method may be preferred to the in vivo method because
◦ It allows the quantification of the response over a wider area of the
◦ Large number of samples can be tested at any one time
◦ The time required for a response to occur is shorter (2-3 days)