Name: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells (Video) | JoVE Journal
Uploaded: 2010-10-27T17:35:00.000+00:00
Duration: 14 min 2 s
Description: Learn to isolate and culture avian embryonic valvular progenitor cells using HH14 endocardial and HH25 mesenchymal cells. This method enables 3D in vitro modeling of valvular morphogenesis for cardiac developmental biology research.

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5 minutes

This research is supported by The Hartwell Foundation, the American Heart Association (Scientist Development Grant #0830384N) and the Foundation Leducq: Mitral 07CVD04.

1. Preparation
<ol>
	<li>Incubate fertile quail or chicken eggs to stage 14- (about 2 days) or 25 (about 4.5 days) at 60% humidity and 37°C.</li>
	<li>Prepare sterile Earl's Balanced Salt Solution (EBSS)
 <ol>
		<li>Add 100 mL 10X EBSS to 600 mL 18 MΩ water</li>
		<li>Add 2.2 g sodium bicarbonate</li>
		<li>Adjust the pH of the solution to 7.2</li>
		<li>Bring the solution up to 1000 mL</li>
		<li>Sterilize the solution by passing through a 0.2 μm filter</li>
 </ol>
 </li>
	<li>Prepare sterile M199 culture medium.
 <ol>
		<li>Add 1 package of M199 powder to 700 mL 18 MΩ water.</li>
		<li>Add 2.2 g sodium bicarbonate</li>
		<li>Adjust the pH of the solution to 7.2.</li>
		<li>Add 10 mL (1%) Penicillin-Streptomycin</li>
		<li>Bring the solution up to 1000 mL</li>
		<li>Sterilize the solution by passing through a 0.2 μm filter</li>
		<li>Add 1 mL sterile 100X Insulin-Transferrin-Selenium-G supplement</li>
		<li>Add 10 mL (1%) sterile chicken serum</li>
 </ol>
 </li>
	<li>Prepare three dimensional collagen gels at a collagen concentration of 1.5 mg/mL. Previous work in our lab has shown that a collagen concentration of more than 1.5 mg/mL provides a matrix that is too biomechanically stiff for the cells to move through. Collagen gel concentrations lower than 1.5 mg/mL have so few collagen fibers that the cell clump can shred the fibers locally, producing an island of cells.
 <ol>
		<li>Add the following ice-cold reagents to a sterile 15 mL centrifuge tube in order:
 	<ul>
 	<li>3X M199: Volume = total volume necessary / 3</li>
 	<li>Sterile 18 MΩ water: Volume = total volume necessary (M199 volume + chicken serum volume + 0.1 M NaOH volume + collagen volume)</li>
 	<li>Sterile chicken serum: Volume = total volume necessary * 0.01</li>
 	<li>Rat tail collagen I: Volume = (collagen gel concentration * total volume necessary) / collagen stock concentration</li>
 	<li>Sterile 0.1 M NaOH: Volume = collagen volume * 0.2 Note: The amount of water, 0.1 M NaOH, and collagen added will vary depending on the concentration of the collagen stock solution.</li>
 </ul>
 </li>
		<li>Mix the collagen solution well with a sterile pipette.</li>
		<li>Immediately transfer 0.3 mL of the collagen solution into 1.9 cm2 growth area wells. This is enough collagen solution to completely cover the well and to provide a gel that is approximately 3 mm thick. A gel thickness of at least 250 μm is necessary for cell invasion studies. </li>
		<li>Allow the collagen solution to gel for at least 1 hour at 37°C, 5% CO2, and 100% humidity.</li>
 </ol>
 </li>
</ol>
2. Pre-EMT Endocardial Cell Isolation and Culture
<ol>
	<li>Embryo isolation
	<ol>
 	<li>Place 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. </li>
		<li>Gently puncture the taped area of the egg in the air cell with the tip of sterilized curved scissors.</li>
		<li>Cut a hole in the taped portion of the egg that is large enough to allow the yolk to pass through. Start the hole at the puncture and cut outward in a spiral shape. Make sure to cut through the outer membrane, but avoid piercing the yolk. </li>
		<li>When the hole in the shell is complete, visualize the quail embryo. The embryo is normally located on the top side of the yolk.</li>
		<li>Begin to pour the yolk through the hole cut in the egg with one hand. Hold a #55 tweezer in the other hand. As the embryo exits the egg on the top side of the yolk, gently slide the #55 tweezers under the embryo and remove it from the yolk. 
Note: For chicken eggs, the embryo isolation involves cracking the chicken egg on a sharp, clean surface and breaking the egg into a 100 mm Petri dish. The embryo should be located on the top side of the yolk. Gently slide #55 tweezers under the embryo and remove it from the yolk.</li>
		<li>Place the embryo into a 100 mm Petri dish containing ice-cold, sterile EBSS.</li>
 </ol>
 </li>
	<li>Stage the embryos according to the criteria of Hamburger and Hamilton1. Embryos at stage 14- are past stage 13, but not yet at stage 15, with 20-21 somites and a tail bud. A key characteristic of stage 14- embryos is the head angle, which is slightly greater than 90° to the body.</li>
	<li>Remove the hearts from quail embryos at stage 14- with #55 tweezers by cutting transversely where the AV canal and OFT make contact with the thoracic wall. Place the hearts together on one side of the Petri dish.</li>
	<li>Collect the isolated hearts with a sterile transfer pipette and place them into a new 100 mm Petri dish filled with sterile, ice-cold EBSS.</li>
	<li>Cut the OFTs and AV canals transversely from the ventricles with #55 tweezers.</li>
	<li>Section the remaining AV canals and/or outflow tracts (OFTs) longitudinally.</li>
	<li>Set a pipette to a volume of 20 uL. Use this pipette to aspirate six longitudinally-cut AV canals and OFTs. </li>
	<li>Expel the AV canals and OFTs onto the collagen gel. Aspirate any excess EBSS from the surface of the gel.</li>
	<li>Use #55 tweezers to orient the explants so they are flat and so the lumen side faces the collagen gel. Try not to puncture the collagen gel with the tweezers.</li>
	<li>After 2 hours at 37°C and 5% CO2, remove the myocardium from the explants with #55 tweezers. The endocardial cells should be left behind on the surface of the collagen gel. Alternatively, the myocardium can be left on the explant. With the myocardium removed, the endocardial cells with not undergo EMT without intervention. With the myocardium present a subset of endocardial cells will transform into mesenchyme, but the degree of transformation can be modulated with external factors. 
Note: Even if the myocardium will be left on, the explant must be allowed at least 2 hours to attach to the gel before medium is added. </li>
	<li>Add 0.4 mL M199 medium to each well containing explants.</li>
	<li>Culture the cells at 37°C, 5% CO2, and 100% humidity.</li> 
</ol>
3. Post-EMT AV Cushion Mesenchymal Cell Isolation and Culture
<ol>
	<li>Crack the chicken egg on a sharp, clean surface and break the egg into a 100 mm Petri dish. The embryo should be located on the top side of the yolk. Pick up the embryo with #5 tweezers by grabbing the membranes surrounding the embryo and then place the embryo into a 100 mm Petri dish filled with ice-cold, sterile EBSS.</li>
	<li> Stage the embryos according to the criteria of Hamburger and Hamilton1. Features of a stage 25 embryo include distinct elbow and knee joints and slight eye pigmentation. </li>
	<li> Isolate the hearts from all HH25 embryos and place the hearts together on one side of the Petri dish. </li>
	<li>Collect the isolated hearts with a sterile transfer pipette and place them into a new 100 mm Petri dish filled with sterile, ice-cold EBSS.</li>
	<li>Isolate the 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. </li>
	<li>Collect the isolated AVs with a sterile transfer pipette and place them into a new 100 mm Petri dish filled with sterile, ice-cold EBSS.</li>
	<li>Sequentially isolate the cushions from the AV. Make sure there is no myocardium present on the AV cushions. </li>
	<li>Collect the isolated cushions with a sterile transfer pipette and place them into a sterile 15 mL centrifuge tube.</li>
	<li>Spin the cushions to the bottom of the centrifuge tube at 15 xg for 3 minutes.</li>
	<li>Use a sterile 1000 mL tip to remove as much of the EBSS supernatant as possible without disturbing the cushions.</li>
	<li>Add 1 mL of 0.25% Trypsin-EDTA that has been pre-warmed to 37°C. Allow the cushions to incubate in trypsin until they are completely dissolved, which is normally about 3-5 minutes.</li>
	<li>Add 100 μL of chicken serum to the centrifuge tube to quench the trypsin. </li>
	<li>Pellet the cells at 160 xg for 5 minutes. Remove the supernatant with a sterile, 1000 μL tip.</li>
	<li>Resuspend the cells in 2 mL of M199 medium and mix by pipetting. Take 10 μL of the cell suspension to count on hemocytometer and determine the total number of cells that have been isolated</li>
	<li>Determine how many collagen gels will be made based on the number of cells isolated. Cells need to be plated at a density of 2 x 105 cells/mL with a 250 μL gel volume.</li>
	<li>Pellet the cells at 160 xg for 5 minutes. Remove the supernatant with a sterile, 1000 μL tip.</li>
	<li>Using the protocol listed above, add 3X M199, water, chicken serum, collagen, and 0.1 M NaOH, in that order, to the cell pellet. Mix by gentle pipetting.</li>
	<li>Add 250 μL to each 1.9 cm2 growth area well. </li>
	<li>Allow the collagen solution to gel for at least 1 hour at 37°C, 5% CO2, and 100% humidity.</li>
	<li>Add 0.4 mL of culture media and incubate overnight.</li>
	<li>To continue culturing the gels in stress-free conditions, liberate the gels from the sides of the culture well using a sterile 200 μL pipette tip. Mechanically constrained cultures are gels that remain attached to the sides of the culture well.</li>
</ol>
4. Representative Results
<img src="/files/ftp_upload/2159/2159fig1.jpg" alt="Embryonic development comparison; microscope images; embryogenesis stages A and B." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1: Examples of embryos. (A) HH14- quail embryo, and (B) HH25 chicken embryo are shown here.
<img src="/files/ftp_upload/2159/2159fig2.jpg" alt="Cell structure analysis, microscopy image, comparison of sample surface morphology, scale bar 200 μm." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2: HH14- valve endocardial explants after 2 hours of culture. (A) with the myocardium present, or (B) with the myocardium removed.
<img src="/files/ftp_upload/2159/2159fig3.jpg" alt="Cell growth comparison, microscopy image, experiment, cell morphology, controlled condition analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3: HH14- valve endocardial explants. (A) With the myocardium present, many of the cells undergo endocardial to mesenchymal transformation and have a spindle-shaped, mesenchymal phenotype after 48 hours of culture. (B) When the myocardium is removed all of the cells maintain a cobblestone-shaped, endocardial phenotype after 48 hours in culture.
<img src="/files/ftp_upload/2159/2159fig4.jpg" alt="Microscopic image of cell culture; scale 200 microns; experimental data for biological study." data-altupdatedat="1788235205000" data-alt="Figure 4 "> Figure 4: HH25 valve mesenchymal cells in a collagen gel. Cells are rounded immediately after seeding.
<img src="/files/ftp_upload/2159/2159fig5.jpg" alt="Microscope images, cell analysis; A: 200 microns scale, B: close-up view; microscopy study." data-altupdatedat="1788235205000" data-alt="Figure 5 "> Figure 5: HH25 mesenchymal cells in culture. (A) After 48 hours of culture in a constrained collagen gel the HH25 mesenchymal cells are beginning to spread. (B) HH25 cells in a stress-free collagen gel 7 days after seeding and 6 days after liberation have compacted the gel to approximately 50% of the original area.

<ol><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22J+Morphol%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li>
<li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invasion+of+mesenchyme+into+three-dimensional+collagen+gels%3A+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li>
<li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho%2FPI+3-kinase%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li>
</ol>

No conflicts of interest declared.

The method for isolating endocardial cells from stage HH14- hearts originally developed by Runyan and Markwald provides a controlled, in vitro environment to study the factors that initiate and modulate embryonic EMT2. HH14- endocardial explants cultured without myocardium will not undergo EMT without biomechanical or biochemical intervention. If the myocardium is left on the explant it will signal a subset of endocardial cells to undergo mesenchymal transformation, but external ...

<table><tbody><tr><td>Extra fine Bonn scissors, curved</td><td>Fine Science Tools</td><td>14085-08</td><td></td></tr><tr><td>Dumont tweezers #55</td><td>World Precision Instruments, Inc.</td><td>14099</td><td></td></tr><tr><td>Dumont tweezers #5</td><td>World Precision Instruments, Inc.</td><td>14098</td><td></td></tr><tr><td>Sterile transfer pipettes</td><td>Samco Scientific, Thermo Fisher Scientific</td><td>2041S</td><td></td></tr><tr><td>4-well culture plates</td><td>Nalge Nunc international</td><td>176740</td><td></td></tr><tr><td>Sterile disposable filter units, PES, 0.2 &amp;mu;m pore size membrane</td><td>Nalge Nunc international</td><td>566-0020</td><td></td></tr><tr><td>Sterile 15 mL centrifuge tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>Sterile petri dishes, 100 mm x 15 mm</td><td>VWR international</td><td>25384-342</td><td></td></tr><tr><td>Laboratory tape</td><td>VWR international</td><td>89097-920</td><td></td></tr><tr><td>Rat-tail collagen I</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>10X Earl&amp;rsquo;s Balanced Salt Solution</td><td>Quality Biological, Inc.</td><td>119-064-131</td><td></td></tr><tr><td>M199 powder</td><td>Invitrogen</td><td>31100-035</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium-G supplement (100X)</td><td>Invitrogen</td><td>41400-045</td><td></td></tr><tr><td>Chicken serum</td><td>Invitrogen</td><td>16110-082</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Sodium hydroxide solution, 1.0 N</td><td>Sigma-Aldrich</td><td>S2770</td><td></td></tr><tr><td>Fertile quail eggs (&lt;em&gt;Coturnix coturnix&lt;/em&gt;)</td><td>Lake Cumberland Game Bird Farm</td><td></td><td></td></tr><tr><td>Fertile chicken eggs (&lt;em&gt;Gallus gallus&lt;/em&gt;)</td><td>Cornell University Poultry Farm</td><td></td><td></td></tr></tbody></table>

isolation and culture of avian embryonic valvular progenitor cells

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

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.

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

Research

JoVE Journal

Biology

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture.  This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

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

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC).  VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolation of Valvular Endothelial Cells

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolation of Embryonic Ventricular Endothelial Cells

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease.  Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolation of Murine Valve Endothelial Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Here, we present a detailed visual protocol for executing the left atrial ligation (LAL) model in the avian embryo. The LAL model alters the intracardiac flow, which changes wall shear stress loading, mimicking hypoplastic left heart syndrome. An approach to overcome the challenges of this difficult microsurgery model is presented.

Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development

Learn to isolate and culture avian embryonic valvular progenitor cells using HH14 endocardial and HH25 mesenchymal cells. This method enables 3D in vitro modeling of valvular morphogenesis for cardiac developmental biology research.

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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