An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In the X ovo chicken embryo culture system preparing shell list chick embryo cultures enables investigation of embryogenesis. The procedure starts with obtaining and incubating fertilized chicken eggs at 37.5 degrees Celsius for 72 hours. Hammocks for holding the egg yolks with the growing embryos are prepared.

Next, then egg yolks can be transferred onto hammocks, and embryogenesis continues outside the shell. This technique can be applied for microinjection into vasculature, ultra sonography and microsurgery, and the results can show how embryos develop under normal and perturbed conditions. Hi, my name is Akshay Shaker and I'm a master's student in the cardiovascular developmental bio engineering lab run by Jonathan Butcher in the Department of Biomedical Engineering at Cornell University.

And the main advantage of our technique over previous methods, such as in ovo egg culture, are that it provides full optical access to the embryo as well as ease of transport without damage. Hello, my name is Char Hin. I'm a postdoctoral researcher at the Cardiovascular Developmental Bioengineering Laboratory, Jonathan Chu Lab at the Department of Biomedical Engineering of Cornell University.

This method can help answer key questions in the developmental biology field, such as how heart defects develop during embryogenesis. Before beginning this procedure sterilize all the materials by wiping with 70%ethanol. To begin the procedure, fill three quarters of a nine ounce plastic cup with warm sterile water.

Repeat for as many cups as necessary. A regular nine ounce cup has a top diameter of eight centimeters, which is the size most suitable for the extended culture application presented here. Next, cut a 20 centimeter long piece of cling wrap.

Cover the top of the nine ounce plastic cup filled with warm sterile water with a piece of cling wrap, and use a rubber band to hold the plastic wrap in place. Cling wrap is the most appropriate for this application since it prevents sticking of the yolk, which may result in splitting of the yolk. During transportation, the plastic should be spread so that it touches the top of the water.

Cut the excess plastic around the band and wipe the plastic. With Kim wipes wedded with 70%ethanol, the hammocks are now ready to support the embryos. Fertilized chicken eggs can be stored at 13 degrees Celsius up to five days before incubation.

Without activating development, a red wine cooler can maintain this temperature in order to have the eggs ready for culturing. Activate the development of the embryo by incubating the eggs one site up in a 60%constant humidity incubator with continuous rocking at 37.5 degrees Celsius for 72 hours. When the eggs are ready, remove them from the incubator.

Sterilize the eggs by wiping with Kim wipes weed with 70%ethanol. Lay the eggs horizontally on their sides on an egg carton for one to two minutes for proper positioning of the embryo. Work with six eggs at most at a time since trying to crack more than six eggs at one time.

May result in keeping the exit room temperature too long, which can affect viability. At the end of one to two minutes, the embryo has slid to the top of the egg. To transfer each embryo to a hammock, work aseptically in a laminar flow hood.

First, crack the underside of the egg gently using a sharp edge like the edge of a metal bucket or a glass beaker. Remember that the embryo is on the upper side. Separate the two halves of the egg shell gently by pulling to the sides with two thumbs.

Then place the embryo, yolk and albumin onto the hammock. The albumin provides a dampening environment around the yolk absorbing shocks caused by moving the system. The albumin also provides nutrition to the embryo during the long culture period.

If the transfer of the embryo is performed properly, the embryo should already be positioned on top. If the embryo is at the bottom, use closed curve, small scissors, or another similar blunt instrument to rub the yolk so that it turns around itself and the embryo moves to the top. If culturing embryos beyond embryonic, day seven, scatter crushed eggshell pieces around the periphery of the embryo as a calcium source for bone development and maturation.

After the transfer of the embryo is complete, place a 10 centimeter diameter Petri dish on top of the cup to seal the embryo. Place the hammock in the incubator set to 37.5 degrees Celsius. If a portable incubator is used, place one to two nine ounce cups filled with water in the incubator as well.

In order to keep the humidity at approximately 60%the embryos continue to develop and can be used for microinjection in microsurgical applications. This embryo culture is suitable for microinjection applications where solutions need to be injected to vasculature. First, prepare the injection apparatus starting with the needles, using a micro forge fashion, pulled glass capillary tubes into beveled tip microneedles.

The size of the tip depends on the flow rate desired. Connect the microneedle syringe to 0.03 inch inner diameter silicone tubing. Load the syringe tubing with a solution to be injected and connect the syringe microneedle to the tubing.

Place the microneedle onto the micro manipulator and syringe to syringe holder or syringe pump. Once the injection assembly is ready, retrieve the embryos. Horizontal penetration of the needle is required for proper entry of the needle into a blood vessel such as a vital vessel.

The embryo therefore needs to be on the same level as the edges of the cup holding it. In order to raise the embryo, take off the rubber band holding the plastic wrap that supports the embryo. Lift one side of the plastic gently and add some water to the cup without moving the embryo too much.

Then place the rubber band on the plastic again. Now that the embryo is raised, the solution can be injected into an embryonic vessel. Note that when injecting a vital vessel, the size of the microneedle needs to be approximately one 10th the size of the vessel diameter.

To maintain efficient perfusion without causing bleeding, first, pump the solution into the microneedle so that no air is trapped inside. Next, align the tip of the microneedle with a selected vessel for vital vessels. Selecting a for area gives maximum area for targeting the vessel.

Once the injection area is defined, push the needle towards the vessel. The vessel first retracts. Once the needle penetrates start perfusion, the color of the flowing blood changes with perfusion.

If the color change is not seen, the needle may have been pushed through the vessel In that case, slowly retract the microneedle while simultaneously pumping solution until a color change is visible in the vessel. When profusion is complete, gently pull the microneedle out of the vessel. This embryo culture is also suitable for microsurgical applications like left atrial ligation, which is performed in the fourth day of incubation.

First, prepare the knots to be used at 0.5 millimeter diameter from 10 zero nylon surgical suture. Using fine forceps, remove the chorionic and LAN toic membranes from the embryo. Lift the embryo from the back and rotate it vertically so that the left side of the embryo is now on top and accessible.

After that, remove the pericardium over the left atrium. Align the knot over the left atri and tighten it by holding and pulling it from the sides With forceps, the knot should be tied so as to prohibit approximately 75%of the original blood flow to the left side. Cut the edges of the knot with micro scissors and carefully remove and discard excess suture.

Finally, rotate the embryo back to its original orientation with the right side on top. Shown here are embryos at different stages cultured X ovo as just demonstrated a water circulation heater can maintain the environmental conditions for extended times, which may be required. During imaging and microsurgical applications.

The chick embryos cultured X ovo are suitable for microinjection. In this experiment, a fluorescent dye was injected into the vasculature of an X ovo cultured chick embryo. The chick embryos cultured X ovo are suitable for microsurgery.

In this experiment, left atrial ligation procedure was performed on an X ovo cultured chick embryo. Once mastered culturing X ovo chick embryos can be done in about five minutes per embryo. After watching this video, it should have a good understanding on how to create XO chick embryo cultures suitable for imaging and microsurgery applications, and where the transport of cultures are necessary.