The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

This protocol describes the preparation and use of chicken embryos between day five and eight of incubation for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery studies The three methods to take the content out of the egg shell at day five to eight allows its development within the shell and only requires basic lab tools. This model is suitable to investigate different aspects of ultrasound and vascular research like blood flow imaging, drug delivery, and new ultrasound contrast agents and transducers. To prepare a five-day-old embryo, place a five-day incubated fertilized egg into a pre-warmed egg holder with the egg in the same orientation as within the incubator and use the pointy backend of a tweezer tip to make a small indentation on the very top of the egg.

Use the tweezers to make a second indentation on the side of the egg about 2/3 down the length of the egg and use a pair of larger tweezers to remove a small piece of eggshell from the indented area at the top of the egg, taking care that the air sac makes contact with the ambient air without penetrating the shell too deeply. Using a five-milliliter syringe equipped with a 19-gauge needle penetrate the shell through the second indentation with the needle pointing downward and withdraw approximately two milliliters of albumin. When the albumin has been collected, use tape to seal the puncture and dispense the albumin into a weigh boat.

Use the large tweezers to enlarge the small opening on the top of the egg until the embryo and chorioallantoic membrane are fully visible without removing the shell below the chorioallantoic membrane or penetrating the inner membrane. When a sufficient opening has been created, turn the egg 180 degrees within the holder so that the opening at the top of the egg is now facing toward the bottom. After one to two minutes, the embryo will float up, becoming invisible from the bottom.

When the entire embryo and chorioallantoic membrane have disappeared from view and only the yolk is visible, remove the tape from the side opening. The inside of the egg should bulge out of the opening. While holding the bottom of the egg close to the weigh boat in the metal weigh boat holder, use the sharp points of the small tweezers to make a horizontal scratch gently but quickly in the membrane over the entire width of the opening and gently drop the egg contents into the weigh boat.

Check whether the heartbeat is still present to confirm that the embryo is still alive, the chorioallantoic membrane vessels are intact, and there is no blood or yolk leakage. Place the viable embryo at 37 degrees Celsius with regular administration of 30-microliter drops of 37-degrees Celsius PBS to the embryo and chorioallantoic membrane. To harvest a six-to seven-day-old embryo and chorioallantoic membrane, prepare the egg as demonstrated for a five-day-old embryo and use a new five-milliliter syringe equipped with a 19-gauge needle to penetrate the shell through the second indentation with the needle pointing downward to withdraw five to six milliliters of albumin in a single penetration.

When the albumin has been collected, insert the needle of a 10-milliliter syringe containing approximately one milliliter of 37 degrees Celsius PBS, more than the withdrawn volume, into the side of the shell with the needle pointing downward to replace the harvested albumin. Then, quickly reseal the gap with a piece of tape and finish processing the egg as demonstrated. To harvest the embryo and chorioallantoic membrane from an eight-day-old fertilized egg, hold the egg horizontally, use the back end of a pair of large tweezers, and make a small indentation about halfway down the length of the egg.

Make small indentations in a ring pattern 360 degrees around the egg shell space approximately 10 millimeters apart. When all of the indentations have been created, use the pointy end of the tweezers to crack the shell between two of the indentations and completely submerge the egg in 37-degree Celsius PBS for five minutes. At the end of the incubation, move the egg over a weigh boat in the PBS and place both thumbs into the large hole to gently open the egg, which should crack along the small indentations.

When the crack has formed around the egg shell, gently pull the two sides apart, while gently moving the pieces back and forth until the egg contents are separated from the shell and drop the contents into the weigh boat. Slowly raise the weigh boat containing the egg contents from the PBS, slightly tilting the way boat to remove any excess PBS. Then, place the weigh boat into a metal weighing boat holder.

At this stage, a beating heart can be seen in the embryo. To prepare an embryo and chorioallantoic membrane sample for microscopy imaging, place a holder with an acoustic membrane with agarose onto the bottom of a one-litter Petri dish containing approximately 500 milliliters of 37-degree Celsius PBS with the agarose layer facing up. Use small scissors to quickly cut into the vitellus membrane in six to seven cuts around the entire chorioallantoic membrane while rotating the weigh boat for better better precision and speed.

Use a tablespoon to scoop up the cutout membrane containing the embryo and chorioallantoic membrane and slowly raise the spoon to allow visual inspection of whether the cut-out membrane containing the embryo and chorioallantoic membrane is still attached to the remainder of the yolk sac membrane. Slightly tilt the spoon to get rid of as much yolk as possible without drying out the tissue and submerge the cut-out membrane in the PBS in the one-liter Petri dish. Using small tweezers, grab and gently swirl one edge of the membrane to remove any yolk that is still attached before moving the membrane over the holder with the acoustic membrane.

Use an insect specimen pin to secure the membrane to opposite corners of the holder and avoid piercing the chorioallantoic membrane vessels. When both corners have been secured, lift and slightly tilt the holder to discard most of the PBS and use the small tweezers to stretch and evenly distribute the membrane over the holder to allow the rest of the membrane to be pinned flat to the holder. When the embryo has been secured, place the holder into a microscopy setup at 37 degrees Celsius and place an acoustically and optically transparent membrane over the region-of-interest on the sample to allow the optical visualization of the vessels.

In this representative analysis, five, six, seven, and eight-day-old embryos and chorioallantoic membranes were removed as demonstrated. No bleeding or damage to the embryos or chorioallantoic membranes can be observed, indicating that these methods can be used to safely harvest the egg content without harming the embryo or the chorioallantoic membrane vessels. Multiple complications can occur during the extraction, so it is important to follow the protocol carefully and to discard any damaged tissue samples.

As demonstrated, the embryo can be injected with, for example, ultrasound contrast agents such as microbubbles. Upon delivery, the micro bubbles can be observed circulating within the lumen of the injected blood vessel and circulating within the peripheral blood for several hours. Before microbubble injection, contrast can already be observed inside the embryonic heart by B mode, but not high-frequency ultrasound sub-harmonic imaging.

The addition of microbubbles increases the contrast and visibility of the embryo in both types of images. High-frequency 3D sub-harmonic images can also be obtained after contrast agent injection to visualize vessel networks within the embryo and chorioallantoic membrane. The chicken embryo and chorioallantoic membrane vessels can also be used to investigate ultrasound-mediated drug delivery.

When attempting this procedure, it is most important to always keep the embryo and CAM at 37 degrees Celsius and to not let it dry out. Following this procedure, the chicken embryo can be placed in different setups, such as a water tank or microscope to perform contrast enhanced ultrasound imaging or microbubble-mediated drug delivery studies. This method and preparation is suitable for ultrasound contrast agent studies and can also be used for cancer and virus research.