Ex ovo CAM assay protocol

Gözde Eke
Naside Mangir

21 Nov 2015
Material(s):

Chemicals:
 1% Penicillin, Streptomycin in PBS
 IMS (20%)
 IMS (70%)
Apparatus:
 10x 10 cm Weighting boat (100 mL)
 Laminar Flow Cabinet
 Rotating Egg Incubator
 Sterile incubator 37.5 °C with 60- 80% humidity (CO2 and O2 supply not
necessary)
 A tissue hot plate (slide dryer)

Procedure:

Preparation of the eggs

1. Receive the eggs laid on the same day of dispatch
2. Clean dirt, feathers and excrement from the egg shells with IMS (20%) sprayed
tissue.
3. Eggs can be kept at +10oC for up to 7- 10 days after arrival.
Incubation of the eggs
1. Day 0: Place the eggs in the Automatic Egg Incubator horizontally. Incubate the
eggs at 37.5oC and 60% humidity while rotating 12 times a day (The incubator does
this automatically)
2. Mark the upper surface of the horizontally lying eggshells with permanent pen to
spot the embryo since the embryo resists rotation to a certain extent.
Ex ovo culture
At day 3, under laminar flow,
1. Sterilize the weighing boats by dipping them in a bath of 70% IMS and leave
them to dry
2. Put 2 mL of PBS+ Pen/Strep into each weighting boat. Remove the eggs from the
rotating incubator and place them horizontally in cartoon egg tracks with the
marked line always facing upwards.
3. Wipe your gloves with 70% IMS.
4. Crack each egg by hitting it to the edge of a 500 mL beaker from below holding it
horizontally with the pen mark on top.
5. Transfer the chick embryo by gently separating to halves of the egg shell to the
sides and upwards. Keep the egg close to the bottom of the weighting boat to
maintain a vacuuming effect between the egg contents and the weighing boat
provided by the egg white.
6. After embryo is transferred to the weighing boat, check and make sure that the
egg yolk is intact without any damage and the chick embryo is alive (observe the
beating heart)
7. Put a sterile petri dish lid to cover the weighing boat and place the weighing boat
on the hot plate at 37.5oC until you transfer them in the incubator
 My survival rate increased from 70% to 80% after I started using a hot plate
to keep the eggs as warm as possible whenever they were not in an incubator.

8. Crack 4-10 eggs at a time to decrease the time when embryos were outside an
incubator.
9. Keep the ex ovo cultures into a 37.5oC stationary sterile incubator with 60%
humidity at the rest of the experiment.
10. During incubation, check daily if;
a. The chick embryos are alive or dead and keep a record of it.
b. The chick embryos are developing properly in accordance with their
chronological age (chronological age [HH]= developmental age[days]).
 Check Hamburger& Hamilton paper
 This will allow you to calculate your survival rate, improve your technique,
monitor any factor that might affect your experiment and also prove that your
experiment groups were comparable with each other.
 Discard the dead embryos immediately according to laboratory guidelines (for
animal waste) to avoid nasty smells and infections.
 Chronological age: 3 days Chronological age: 3 days
Structural/ morphological age: HH 14 Structural/ morphological age: HH 19

Figure 1. An example of an inconsistency between the chronological age and the morphological age of
the chick embryos that died at day 3. Although it can be hard for non- experts to determine exact
developmental age, it is better to observe the most obvious inconsistencies. (Arrows show the limb bud
and the eye of the embryo on the right which have not yet developed in the embryo on the left)

Application of test samples

On day 8, introduce the test sample (hydrogels, electrospun fibers with or without cells,
growth factors or drugs) into ex ovo cultures.
 The samples should ideally be placed halfway between the embryo and the outer
border of the CAM and in between two large vessels (Figure 2).
 It is very important to use identical samples in terms of size and shape because
the evaluation of results will mainly be based on the quality of the pictures.
Figure 2. Proper placement of a test sample on the CAM at day 8 of embryonic development. Arrow
shows the test sample and the dashed line the border of the CAM. Also note that the sample was
placed in between two large vessels.

Depending on the test material used and the purpose of the experiment obtain images
between days 10- 14 and end the experiment accordingly.
 It might be useful to take daily images between days 10- 14 initially to find out
which time point gives you the best results.

Before taking images at the end of your experiment make sure once again that the
embryos are alive as dead embryos will result in false negative results (Figure 3).

To improve the quality of the image a colored liquid (a 20% emulsion or methylene blue)
can be injected just beneath the test sample and the CAM which can conceal the
unnecessary background increase the contrast (Figure 4).

Sample retrieval
Retrieve the tested samples with 0.5 mm margin by spring scissors.
Place the samples into paraformaldehyde (3.7%).
Sacrifice the chick embryos by cutting the vitelline artery with spring scissors
immediately after retrieving the samples.
 DEAD DEAD ALIVE

Figure 3. The difference between the CAM of the embryos that are dead or alive. The CAM of a healthy
embryo is bright, glossy, moist and even the smallest vessels contain some blood whereas the blood vessels of
the dead embryos are barely visible as there is no circulation at all.

Before injection After injection

Figure 4. Comparison of the pictures taken before and after a coloured substance injected beneath the
CAM. The images belong to electrospun PLA on CAM at day 14. A 20% emulsion and a methylene
blue solution was injected on the upper right and lower right, respectively.
Useful reading:

1. Hamburger V, Hamilton HL. A series of normal stages in the development of

the chick embryo. Journal of morphology. 1951;88(1):49-92.

2. Zudaire E, Cuttitta F. The Textbook of Angiogenesis and Lymphangiogenesis:

Methods and Applications: Methods and Applications: Springer Science &
Business Media; 2012.

3. Ribatti D, Nico B, Vacca A, Presta M. The gelatin sponge chorioallantoic

membrane assay. NATURE PROTOCOLS-ELECTRONIC EDITION-.
2006;1(1):85.