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&deg;C.</li>
	<li>Prepare sterile Earl's Balanced Salt Solution (EBSS)
 <ol>
		<li>Add 100 mL 10X EBSS to 600 mL 18 M&Omega; 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 &mu;m filter</li>
 </ol>
 </li>
	<li>Prepare sterile M199 culture medium.
 <ol>
		<li>Add 1 package of M199 powder to 700 mL 18 M&Omega; 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 &mu;m 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 M&Omega; 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 &mu;m is necessary for cell invasion studies. </li>
		<li>Allow the collagen solution to gel for at least 1 hour at 37&deg;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&deg; 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&deg;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&deg;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&deg;C. Allow the cushions to incubate in trypsin until they are completely dissolved, which is normally about 3-5 minutes.</li>
	<li>Add 100 &mu;L 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 &mu;L tip.</li>
	<li>Resuspend the cells in 2 mL of M199 medium and mix by pipetting. Take 10 &mu;L 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 &mu;L gel volume.</li>
	<li>Pellet the cells at 160 xg for 5 minutes. Remove the supernatant with a sterile, 1000 &mu;L 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 &mu;L to each 1.9 cm2 growth area well. </li>
	<li>Allow the collagen solution to gel for at least 1 hour at 37&deg;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 &mu;L 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="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="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="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="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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">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 ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
<tr>
<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
<tr>
<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
<tr>
<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
<tr>
<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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 ...

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

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Biology

10.1K ビュー.  Cornell University. この記事では、単離するための方法および培養ウズラやニワトリHH14を提供します -弁心内膜細胞とHH25バルブクッション間葉系細胞。

ビデオ: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

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.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular 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.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...