Hi, I'm Susan Paa. I'm from the Institute of Physiological Chemistry, department of Biochemical Endocrinology at the University of Burg Esen in Germany. Hi, I'm Daniel Ze Basian Dole.
I'm from the same institution as Susan. Hi, I'm Mann from the Institute for Anatomy Department of Neuro at the University of Burg. We are going to show you how CH X culture and co membrane essay really work.
So let's go to the lab. The, in most of the recent experimental studies, the cam that is the Chico membrane, was exposed by cutting a window through the eggshell, and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and in the possibilities for observation and photo documentation of effects. X ovo cultures of chick embryos significantly facilitate the accessibility of the cam and the chick embryo.
Please see the beating heart enabling easy in vivo documentation of effects and experimental manipulation of the embryo. In our video article, we are going to provide a step-by-step demonstration of the successful application of X ovo culture for large number of Scientific questions. The eggs are incubated In an incubator with a moving tray.
Here you can see the heating element and the tray mover rotating the eggs 12 times a day continuously. The humidity is kept at 60%by adding water to plastic containers at the bottom of the incubator, and the incubation temperature is set at 37.5 degrees centigrade before starting incubation. Dirt, feathers and excrements are carefully removed from the eggshells, mechanically wiping the eggs with ethanol or disinfectant, however, significantly reduce the survival rates of the embryos.
The eggs are positioned horizontally on the moving tray of the incubator. Please make sure to keep enough distance between the individual eggs to enable free Rotation. After incubation For 72 hours, the eggs are removed from the incubator and the top side of the eggs where the embryo resides is marked by pencil.
Since the embryo resists rotation to a certain extent and remains located here For a better feeling, No gloves should be used to crack the eggs, but the hands should be disinfected with 70%ethanol. The egg is held horizontally with a pencil mark on top and cracked on the edge of a triangular magnetic stirring bar For maximum force control during the cracking procedures, the elbows should not rest on the bench top. It is important That the eggshell as well as the egg membrane are opened during cracking leaking of egg white is a safe sign that the egg membrane has been perforated.
The egg is kept close to the bottom of the patriot dish. To avoid further leakage of egg white during this initial opening procedure, it is important that the egg white on the bottom of the patriot dish is still connected to the rest inside of the egg. As this results in a vacuum inside the egg holding the yolk in place by gently applying pressure to the meridian running through the crack and the equator of the egg by thumb and middle fingers, it is possible to regulate the vacuum and the extrusion of the egg contents.
The content of the egg can then be transferred to the Petri dish with the yolk undamaged. If the egg is gently lifted and both halves of the shell are carefully separated, The ex ovo cultures Are then placed on a tray, returned to an incubator and kept at 37.5 to 38.2 degrees centigrade and 70%humidity. A carbon dioxide or oxygen supply is not necessary.
If special patriot dishes with spacers between lid and dish and incubators with a ventilation grid are used, the ventilation grid should be kept halfway Open. Autoclave filter Papers are used as supporting material for liquid substances applied to the cam as they seem to cause the least irritation of the cam. The application site should be halfway between the embryo and the border of the cam, as otherwise the embryo hinders the microscopic observation of effects in transmitted light.
The liquid here atropin should be applied immediately after the filter has been placed onto the cam to avoid drying. Either additional doses or buffer should be reapplied every day. When the cam is fully developed up to six different samples may be applied and there effects can be compared on a Single cam rings cut From a one milliliter pipette tip should be cut as thin as possible to minimize weight and to avoid indentation of the cam.
The ring is then applied to the cam and the cells are pipetted into The ring. A Hamilton Microliter syringe is rinsed several times with 70%ethanol and finally with sterile phosphate buffered saline or PBS, The micro syringe is filled with a prepared cell suspension carefully But quickly penetrate the cam and the eye layers with the syringe needle and continuously inject the cell suspension. The needle should remain some seconds in the eye to avoid a loss of the injected cell suspension By leakage check X Ovo.
Cultures of eggs incubated for three days are used as limb bud donors. The embryo is dissected from the connected yolk vessels. By cutting A circle, The embryo is transferred to a Petri dish filled with cold sterile PBS by use of a small drain spoon and washed by agitation.
The embryo is then transferred to a fresh Petri dish filled with sterile PBS and freed from the surrounding membranes, carefully tearing them apart with a fine forceps. The limb buds are detached by forceps as close to the body as possible, grasped and transferred to the cam of an eight to 10 day old host chicken. At the desired application site, the cam is selectively traumatized by gentle scraping with one chunk of the forceps.
Then the graft is grasped and layered onto the cam with the other chunk of the forceps. The sacrifice female Mouse is fixed on a board. The abdominal wall is moistened with 70%ethanol cut along the midline and the skin flaps are fixed laterally By pins.
The Uterus are removed from the abdomen, detached and transferred to a beaker. With cold PBS. The embryonic membranes are removed carefully by the use of forceps for demonstration purposes.
A 16 day old mouse embryo is shown here for better visibility of the limb buds. Optimal grafting results are only obtained. However, if limb buds from 10 to 13 day old embryos are used, the limb buds are cut off or the use of fine forceps as close to the body as Possible.
At The desired application site, the CAM is selectively traumatized by gentle scraping with forceps. Then the limb buds are grasped and transferred to the CAM of an eight to 10 day old host chicken. The graft is placed once or twice on a side of the cam where no final grafting is intended to remove excess PBS.
The grafts are ideally placed on the cam near a Y bifurcation of a blood vessel tumor. Biopsies are cut into one to two cubic millimeter pieces by sterile scalpels. Taking material from the surface of the biopsy increases the risk of contamination with muscle or connective tissue.
At the desired application site, the CAM is selectively traumatized by gentle scraping. A piece from the core of the biopsy sample is grafted onto the CAM of an eight to 10 day old host chicken. The grafts are ideally placed on the cam near a Y bifurcation of a blood vessel.
The graft is then transferred to the CAM With minimal PBS attached. Light pressure might be applied to maneuver the graft into a resulting indentation of the cam. The host Chick embryo is killed by decapitation.
The CAM with the attached graft is removed by a circular cut and transferred to a Petri dish filled with PBS. Excessive CAM is cut from the graft except for the former attachment site. Again At the desired application site.
The cam of a new host chick embryo is selectively traumatized by gentle scraping and the graft is transferred to the cam of the second host with minimal PBS Attached. We Just Demonstrated how CH X or culture really works. I showed you the advantages compared to in Orval experiments and gave you some examples for its application.
So that's it. Thank you for watching and good luck with your Experiments.
