Hi, I'm Joel Glover from the Norwegian Center for Stem Cell Research. An important issue in stem cell research is the design of in vivo systems for testing the differentiation potential of stem cells. In this video, my colleagues and I will demonstrate how to use the chicken embryo as an in vivo host for xeno transplantation.
Studies of human stem cells, fertilized eggs are allowed to develop in a warm humidified incubator until the embryos have reached the desired stage. First, wash the eggs with 70%ethanol, Then remove about four milliliters of albumin with a syringe and an 18 gauge needle to create an air pocket over the embryo. Avoid piercing the yolk.
During this process, the air pocket is necessary to avoid harming the embryo. When opening the window in the eggshell seal, the hole made by the needle with conventional plastic tape. The next step is to cut a window through the eggshell over the air pocket to strengthen the eggshell and to prevent eggshell particles from falling onto the embryo.
Place tape over the air pocket and then cut through this using a sturdy dissection, scissors staying within the bounds of the air pocket. Any air bubbles present over the embryo can be popped with a blunt probe like the back end of a pipette tip. To contrast the embryo, prepare a 0.1%fast green solution in chicken physiological saline and filter this through a 0.22 micron millipore filter.
Bend a 25 gauge needle and draw the fast green solution into a one milliliter syringe. To inject the dye under the embryo without damaging the embryonic plate, insert the needle through the wall of the egg yolk outside of the plate. Here you can see the fast green solution spread beneath the transparent embryo, which then presents as a white ghost against the dark background to gain access to the embryo.
First cut and deflect the villin membrane covering the region of interest using a sharp tungsten needle, then use a fresh fire etched tungsten needle to excise a piece of the neural tube unilaterally. Use sharp upward strokes. Typically, it is best to make one or both of the longitudinal cuts before making transverse cuts.
Remove excess tissue with a glass micro pipette and mouth suction. Draw a small volume of the cells to be injected into the tip of a glass micro pipette connected to plastic tubing. Mount the pipette in a micro pipette holder, fitted for pressure injection and connected to an air driven micro pump.
Using a micro manipulator position the micro pipette tip and deposit the cells carefully into the lesion by slowly increasing the pressure To make lesions in the hind brain. Start by removing the vilin membrane as shown earlier. Excise a piece of the hind brainin neural tube unilaterally with a sharpened tungsten needle.
Excess tissue can be removed with a glass micropipet using mouth suction. Then Inject the cells into the lesion as shown earlier. For spinal cord lesions, cells can also be injected directly into the neural tube lumen.
For this, a sharp cell filled micro pipette is used to pierce through the neural tube wall, and then the cells are injected by slowly increasing the pressure. Stem Cells can also be delivered by intravascular injection into extra embryonic vessels, for example, into a vein. As shown here, the cells are mixed with fast green dye to more clearly show their movement within the circulatory system.
Note the rapid spread to the heart and thereby to the embryonic circulation. Here, an injection into an extra embryonic artery. In both cases, a sharp micro pipette must be jabbed sharply through the vessel wall, and then retracted slightly to release the resistance before injecting the cells following injection.
Do not move the egg for several minutes to allow the injected cells to settle and adhere to the host tissue. During this time, the window can be covered with damp tissue paper, then seal the window tightly using plastic tape. Make sure that the albumin cannot leak between the tape and the eggshell.
This is almost always fatal during later incubation. Here We can see the embryo through the window in the eggshell several days after the injection of cells. Note the typical embryonic movements to remove the embryo from the egg.
First, fill a small bowl with ice cold phosphate buffered saline. Then cut through the tape around the window and gently crack the egg releasing the contents into the bowl. The cold temperature acts as an anesthetic through hypothermia.
Cut the extra embryonic membranes around the embryo and then transfer the embryo to a silicone floor dissection containing ice cold physiological saline. Gently remove the extra embryonic membranes, then decapitate the embryo And remove the heart. After pinning the embryo dorsal side down, the remaining visceral can be removed To reveal the ventral surface of the vertebral column.
Excess blood should Be flushed away with a pipette carefully cut between the vertebrae and the spinal cord in two parallel longitudinal cuts and remove the vertebrae to reveal the ventral surface of the spinal cord. The spinal cord can then be dissected free by cutting through the nascent ventral and dorsal roots on either side. We hope this video was useful to you and we wish you good luck in your experiments.
