In this video, a method for the dissection and culture of mouse embryonic lungs is demonstrated. The procedure begins with the harvesting of embryos at E 11.5 or E 12.5. Next, the embryos are opened and the lungs are extracted.
The lungs are then plated in a liquid air interface and incubated following culture. Changes in the growth and branching of mesen chy and epithelium of the embryonic lung in response to specific treatments can be observed using a stereo microscope. Hello, I'm David War Burton.
I direct the developmental biology and regenerative medicine program at the Ban Research Institute Children's Hospital, Los Angeles. And I'm Jan Carro, a senior postdoc in Dr.War Burton Lab. Today we are going to show you a technique for isolating embryonic mouse lung and placing them in organ culture.
This technique has evolved from the original description by Tina Jaco Halkin and Pablo bring us at the USC School of Dentistry about 20 years ago. We use this procedure in our lab to study lamb branching morphy. So let's get started.
On Postum Day 11.5 or 12.5, place a carbon dioxide ssis sacrificed timed pregnant mouse under a laminar flow hood on its back. Spray with 70%ethanol. Make an incision in the abdomen.
Then while holding the animal's hind legs, remove the skin by pulling upwards. Open the peritoneal cavity and remove the uterus. Place the uterus in a 50 milliliter conical tube filled with cold hanks balanced salt solution or HBSS and gently rock for two minutes to rinse off the blood.
Next, to transfer the uterus to a Petri dish under a stereoscopic dissecting microscope. Using spring scissors, cut the uterine wall to release the embryos. Then using a perforated spoon, harvest the embryos and place them in a Petri dish containing HBSS and place on ice.
Continue working under the stereoscopic dissecting microscope and begin dissecting embryonic lungs. Begin by placing the embryo on its left side. Using forceps, remove the right forelimb holding forceps in the left hand.
Cut the embryo starting from the limb opening and remove the skin. This step will allow you to see internal organs and to localize the heart and lungs for the following step. Remove the heart.
The embryonic lungs are located posterior to the heart and anterior to the spine. Next, separate the intact lung from the embryo. Then trim away extraneous tissue and the esophagus.
Once the lungs have been removed, use a sterile transfer pipette to transfer them to a Petri dish containing cold HBSS on ice. To each well of a no clone delta polystyrene dish at 0.5 to one milliliter of D-M-E-M-F 12 supplemented with 50 units per milliliter of penicillin streptomycin. Next place a nucleo polycarbonate track etch membrane on top of the media, shiny side down and rough side up.
Using a sterile transfer pipette place a dissected embryonic lung on top of the nucleo polycarbonate track etch membrane. Using the stereo microscope, adjust the embryonic lung position. The embryonic lung should have an intact trachea placed in a straight position as shown here.
This ensures that all of the lungs have the same internal pressure, transfer the dish to a cell incubator and culture for the desired time. Here, E 12.5 lungs were grown in the presence or absence of FGF nine. FGF nine is a member of the fibroblast growth factor family that induces expansion of the mechy and the dilation of the epithelium in lungs grown in vitro after 48 hours.
In the absence of FGF nine, increased branching is seen. These images are photographs of the embryonic lung taken under a stereo microscope. In contrast, lungs grown 48 hours in the presence of FGF nine have dilated epithelium and mechy.
After 24 hours of culture. After 48 hours, the effect on the epithelium is even more pronounced. So we have just shown you how to prepare organ cultures from embryonic mouse lung.
When doing this procedure is important to remember to remove the the lung from the embryo as soon as possible to reduce the tissue deterioration. So thanks for watching. Hopefully you'll find this a useful technique.
Good luck with your experiments.
