In order to isolate in culture, avian valvular HH 14 minus endocardial cells or HH 25 cushion mesenchymal cells. Fertile legs are incubated for two days. First stage HH 14 minus or 4.5 days for HH 25.
The embryos are then isolated and the heart's removed. The next step of the procedure is isolation of the HH 14 minus atrial ventricular canals and outflow tracks, or the HH 25 atrial ventricular canals. The HH 14 minus atrial ventricular canals and outflow tracks are cut longitudinally or the HH 25 atrial ventricular valve cushions are isolated.
The HH 14 minus x explants are placed lumen side down onto collagen gels. The HH 25 valve cushions are digested with trypsin and the cells seeded within collagen gels and both are cultured. Ultimately, these 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valv agenesis.
Hi, my name is Gretchen Mahler and I'm in the biomedical engineering department at Cornell University. And the methods you're gonna learn today are going to help you answer key questions in the cardiac developmental biology field, such as what factors can initiate and modulate embryonic. Valvular EMT.
Hey, my name's Russell Gould. I'm here at Cornell University in the biomedical engineering department working in the butcher lab. Before we get started today, it's important to remember that visualization of these techniques are key as the isolation steps are difficult to learn.
This is due to the evolving geometry of the embryonic chick heart and the small scale that these forceps are used with. So let's get started. To begin this procedure, prepare sterile Earl's balanced salt solution, abbreviated EBSS and sterile M1 99 culture medium as detailed in the accompanying written protocol.
Next, prepare three dimensional collagen gels by adding the following ice cold reagents to a sterile 15 milliliter centrifuge tube. In this order, three XM 1 99 sterile, 18 mega water, sterile chicken serum rat tail collagen one and sterile 0.1 molar sodium hydroxide. The required collagen concentration is 1.5 milligrams per milliliter.
A higher concentration provides a matrix that is two mechanically stiff for the cells to move through. Lower concentrations have so few collagen fibers that the cell clump can shred the fibers locally producing an island of cells. Mix the collagen solution well with a sterile pipette and immediately transfer 0.3 milliliters of solution into 1.9 square centimeter growth area wells.
This is enough collagen solution to completely cover the well and to provide a gel that is approximately three millimeters thick. A gel thickness of at least 250 micrometers is necessary for cell invasion studies. Allow the collagen solution to gel for at least one hour at 37 degrees Celsius, 5%carbon dioxide and 100%humidity before proceeding to isolate embryonic heart cells, incubate fertile chicken or quail eggs to stage 14 minus at 60%humidity in 37 degrees Celsius.
For quail eggs, place a one inch long piece of laboratory tape over the larger end of the egg. This is where the air cell is located. The tape stabilizes the fragile quail egg shell with the tip of sterilized curved scissors.
Gently puncture the taped area of the egg in the air cell. Cut a hole in the taped portion of the egg that is large enough to allow the yolk to pass through. Start the hole with the puncture and cut outward in a spiral shape.
Make sure to cut through the outer membrane, but avoid piercing the yolk when the hole in the shell is complete. Visualize the quail embryo. The embryo is normally located on the top of the yolk.
Begin to pour the yolk through the hole, cutting the egg with one hand hold number 55 tweezers in the other hand as the embryo exits the egg on the top of the yolk gently slide the number 55 tweezers under the embryo and remove it from the oak. Place the embryo into a 100 millimeter Petri dish containing ice. Cold, sterile EBSS.
Stage the embryos according to the criteria of hamburger and Hamilton or HH embryos at stage 14 minus are past stage 13, but not yet At stage 14 with 20 to 21 somites and a tail bud. A key characteristic of stage 14 minus embryos is the head angle, which is slightly greater than 90 degrees to the body. After isolating the embryos, use number 55 tweezers to remove the hearts by cutting transversely where the AV canal and OFT may contact with the thoracic wall.
Place the hearts together on one side of the Petri dish. Collect the isolated hearts with a sterile transfer pipette and place them into a new 100 millimeter Petri dish filled with sterile ice cold EBSS. Next, use number 55 tweezers to cut the outflow tracks abbreviated LFTs and the atrial ventricular or AV canals transversely from the ventricles section, the remaining AV canals or TS longitudinally.
Set a pipette to a volume of 20 microliters. Use this pipette to aspirate four longitudinally cut explan expel the explan onto the collagen gel. Aspirate any excess EVSS from the surface of the gel.
After transferring the tissue sections, use number 55 tweezers to orient the explan so that they're flat and the lumen side faces the collagen gel. Try not to puncture the collagen gel with the tweezers. Allow the tissue to incubate for two hours at 37 degrees Celsius and 5%carbon dioxide.
After the two hour incubation, one can remove the myocardium from the explants. With number 55 tweezers, the endocardial cells should remain on the surface of the collagen gel. With the myocardium removed, the endocardial cells will not undergo endocardial T mesenchymal transformation or EMT without intervention.
Alternatively, with the myocardium left on the explan, a subset of endocardial cells will transform into mesenchyme, but the degree of transformation can be modulated with external factors. Add 0.4 milliliters of M1 99 medium to each well containing explan and culture. The cells incubate fertile chicken eggs to stage 25 at 60%humidity in 37 degrees Celsius.
Crack the chicken egg on a sharp clean surface and break the egg into a 100 millimeter Petri dish. The embryo should be located on the top of the oak. Pick up the embryo with number five tweezers by grabbing the membrane surrounding the embryo.
Place the embryo into a 100 millimeter Petri dish filled with sterile ice cold. EBSS Stage the embryos according to the HH criteria. Features of an HH stage 25 embryo.
Include distinct elbow and knee joints and slight eye pigmentation. Next, isolate the hearts from all HH 25 embryos and place the hearts together on one side of the Petri dish. Collect the isolated hearts with a sterile transfer pipette and place them into a new 100 millimeter Petri dish filled with sterile ice.Cold.
EBSS isolate the atrial, ventricular or AV region from all hearts at once. Place the AVS together on one side of the Petri dish. Gently agitate the AV region to remove all blood, which can affect cell viability.
Collect the isolated AVS with a sterile transfer pipette and place them into a new hundred millimeter Petri dish filled with sterile ice cold EBSS. Now sequentially isolate the cushions from the avs. Make sure there is no myocardium present on the AV cushions.
Collect the isolated cushions with a sterile transfer pipette and place them into a sterile 15 milliliter centrifuge tube. Spin the cushions to the bottom of the centrifuge tube at 15 G for three minutes At room temperature, use a sterile 1000 microliter tip to remove as much of the EBSS sate as possible without disturbing the cushions. After concentrating the cushions by centrifugation trypsin eyes, the cells for about three to five minutes as detailed in the accompanying written protocol.
All take 10 microliters of the cell suspension to count on a hemo cytometer and determine the total number of cells that has been isolated before transferring the cells to the collagen matrix. Determine how many collagen gels to make based on the number of cells. Isolated cells need to be plated at a density of two times 10 to the fifth cells per milliliter with a 250 microliter gel volume.
In order to combine the cells with a collagen matrix. First pellet the cells at 160 G for five minutes at room temperature. Remove the sate with a sterile 1000 microliter tip, then add ice cold three XM 1 99 water chicken serum collagen and 0.1 molar sodium hydroxide in that order to the cell pellet mixed by gentle pipetting pipette 250 microliters of the cell collagen matrix.
Mix into 1.9 square centimeter growth area wells. Allow the collagen solution to gel for at least one hour at 37 degrees Celsius, 5%carbon dioxide and 100%humidity. Then add 400 microliters of culture media and incubate overnight to continue culturing the gels in stress-free conditions.
Use the sterile 200 pipette tip to liberate the gels from the sides of the culture well. Mechanically constrained cultures result from gels that remain attached to the sides of the culture. Well, an HH 14 minus quail embryo and an HH 25 chicken embryo are shown here.
A key characteristic of stage 14 minus embryos is the head angle at stage 14 minus the head angle is at a slightly greater than 90 degree angle to the body. Features of a stage 25 embryo include distinct elbow and knee joints and slight eye pigmentation. The HH 14 minus valve endocardial implants are shown after two hours of culture with the myocardium present or removed in HH 14 minus valve endocardium explan.
After 48 hours of culture with the myocardium present, many of the cells have undergone endocardial to mesenchymal transformation and now have a spindle shaped mesenchymal phenotype. When the myocardium is removed, all the cells maintain a cobblestone shaped endocardial phenotype. After 48 hours in culture, HH 25 valve mesenchymal cells in a collagen gel are rounded immediately after seeding.
After 48 hours of culture in a constrained collagen gel, the HH 25 mesenchymal cells are beginning to spread HH 25 cells and a stress-free collagen gel. Seven days after seeding and six days after liberation have compacted the gel to approximately 50%of the original area. Well, I hope you learned a lot today.
However, it's important to remember that any experimental results are highly dependent upon the purity of your isolation. So quality should always be precedent over quantity. After watching this video, you should have a good understanding of how to isolate in culture, chicken or quail valve, endocardial cells or mesenchymal cells.
So that's it. Thanks for watching and good luck with your experiments.
