Name: isolation of mouse interstitial valve cells to study the calcification of the aortic valve in vitro
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Description: Watch this Scientific Journal Video about Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro at JoVE.com

NOTE: All animal procedures described here have been approved by Icahn School of Medicine at Mount Sinai institutional core and use committee.
1. Preparation before valve cell isolation from adult mice
<ol>
	<li>Clean and sterilize all the surgical instruments shown in Figure 1A by using 70% v/v ethanol and subsequently autoclaving them for 30 min. clean the surgical workspace with 70% ethanol.</li>
	<li>Add 500 &#181;L of penicillin-streptomycin to 50 mL of 10 ...

<ol>
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G., Uphoff, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+contamination+of+cell+cultures:+Incidence,+sources,+effects,+detection,+elimination,+prevention.">Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.</a> Cytotechnology. 39 (2), 75-90 (2002).</li><li>Richards, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side-specific+endothelial-dependent+regulation+of+aortic+valve+calcification:+interplay+of+hemodynamics+and+nitric+oxide+signaling.">Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling.</a> American Journal of Pathology. 182 (5), 1922-1931 (2013).</li><li>Bouchareb, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+strain+induces+the+production+of+spheroid+mineralized+microparticles+in+the+aortic+valve+through+a+RhoA/ROCK-dependent+mechanism.">Mechanical strain induces the production of spheroid mineralized microparticles in the aortic valve through a RhoA/ROCK-dependent mechanism.</a> Journal of Molecular and Cellular Cardiology. 67, 49-59 (2014).</li><li>Lerman, D. A., Prasad, S., Alotti, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcific+aortic+valve+disease:+molecular+mechanisms+and+therapeutic+approaches.">Calcific aortic valve disease: molecular mechanisms and therapeutic approaches.</a> European Cardiology. 10 (2), 108-112 (2015).</li><li>Janssen, J. W., Helbing, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenazo+III:+an+improvement+of+the+routine+calcium+determination+in+serum.">Arsenazo III: an improvement of the routine calcium determination in serum.</a> European Journal of Clinical Chemistry and Clinical Biochemistry. 29 (3), 197-201 (1991).</li><li>Ortlepp, J. 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This article presents a detailed protocol of mouse valve cell isolation for primary culture. Three aortic valves from 8-week-old mice were pooled to obtain an adequate number of cells. In addition, this protocol describes the characterization of VIC phenotype and the in vitro mineralization assay. The method was adapted from the previously described protocol from Mathieu et al.7.
During the isolation of aortic valves, care must be taken to avoid all so...

Calcified aortic valve disease (CAVD) is the most prevalent valvular heart disease in western populations, affecting nearly 2.5% of elderly individuals over 65 years of age1. CAVD affects over six million Americans and is associated with changes in the mechanical properties of the leaflets that impair normal blood flow-through1,2. Currently, there is no pharmacological treatment to stop the progression of the disease or to activate mineral regression. The only effective therapy to treat CAVD is aortic valve replacement by surgery or transcatheter aortic valve replacement3. It is therefore imperative to investigate the molecular mechanisms leading to valve mineralization to identify new pharmacological targets. Indeed, non-treated aortic stenosis has several adverse consequences such as left ventricle dysfunction and heart failure4.
The aortic valve consists of three layers known as fibrosa, spongiosa, and ventricularis, which contain VICs as the predominant cell type5. The fibrosa and the ventricularis are covered by a layer of vascular endothelial cells (VECs)5. The VECs regulate the permeability of inflammatory cells as well as paracrine signals. Increased mechanical stress may affect the integrity of the VECs and disturb the homeostasis of the aortic valve, leading to inflammatory cell invasion6. Scanning electron microscopy analyses showed disrupted endothelium in a human calcified aortic valve7. 
Histological analyses of calcified tissue reveal the presence of osteoblasts and osteoclasts. Furthermore, osteogenic differentiation of VICs was observed both in vitro and in human valve tissue8. This process is mainly orchestrated by the Runt-related transcription factor 2 (Runx2) and the bone morphogenetic proteins (BMPs)8,9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 mm cutting edge scissors</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle Actin antibody</td>
			<td>abcam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse, Alexa Fluor 488 conjugate</td>
			<td>Cell Signaling</td>
			<td>4412</td>
			<td></td>
		</tr>
		<tr>
			<td>Arsenazo-III reagent set</td>
			<td>POINT SCIENTIFIC</td>
			<td>C7529-500</td>
			<td>a Kit to measure the concentration of calcium</td>
		</tr>
		<tr>
			<td>Bonn Scissors</td>
			<td>F.S.T</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydroxide</td>
			<td>SIGMA -Aldrich 31219</td>
			<td>31219</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Novusbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I&#160; (125 units/mg)</td>
			<td>Thermofisher Scientific</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Tthermofisher</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine graefe forceps</td>
			<td>F.S.T</td>
			<td>11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco 16000044</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>SIGMA-ALDRICH</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M solution</td>
			<td>STEMCELLS TECHNOLOGIES</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100x</td>
			<td>Thermofisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycozap</td>
			<td>Lanza</td>
			<td>VZA-2011</td>
			<td>Mycoplasma elimination reagent</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>SIGMA-ALDRICH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penecillin streptomycin 100x</td>
			<td>Thermofisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100 mM</td>
			<td>Thermofisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>&#160;F.S.T</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>abcam</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mouse interstitial valve cells to study the calcification of the aortic valve in vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

As murine aortic valves are typically 1 mm in diameter, at least three valves must be pooled to collect a million viable cells for different experimental procedures. The different steps of the VIC isolation process are shown in Figure 1 and Figure 2. As it is difficult to manually scrape the valve tissue, it is preferable to use shear stress created by vortexing to remove the VECs. Indeed, the CD31 immunofluorescence staining results showed the absence of endoth...

Watch this Scientific Journal Video about Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro at JoVE.com

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

The use of isolated mouse valve cells is essential to investigate the signaling pathway leading to valve calcification in the use of genetically modified cells from transgenic mice. The two-step digestion is a quick and efficient method. The most challenging part of this protocol is the isolation of the aortic valve from eight weeks mice.It is better to practice on older mice to visualize the aortic valve better. Before starting the experiment, clean and sterilize all the surgical instruments and workspace with 70%ethanol and autoclave the surgical instruments for 30 minutes. When the initial setup is ready, start with cleaning the chest and the abdomen region of an eight week old euthanized mouse using ethanol.Using scissors, open the abdomen and the chest of the mouse, then cut between the left atrium and the left ventricle with small surgical scissors. Remove blood from the heart by perfusing 10 milliliters of cold PBS and cut the heart, keeping three millimeters of the ascending aorta. Under a stereomicroscope, dissect the aortic valve by cutting the heart horizontally in the middle of the ventricles and cutting the left ventricle towards the aorta, then carefully dissect the aortic valve.Pool the valves together in a small 35 millimeter tissue culture dish. Wash the isolated valves in a 75 milliliter cell culture dish with five milliliters of freshly prepared 10 millimolar cold HEPES supplemented with antibiotics and incubate in five milliliters of collagenase type one for 30 minutes at 37 degrees Celsius with continuous shaking. After incubation, centrifuge the tube for five minutes, at 150 times G and wash the pellet once with two milliliters of 10 millimolar HEPES with vortexing at high speed for 30 seconds.Collect the resultant suspension from the tube into a 35 millimeter culture dish and use thin tweezers to carefully transfer the tissue fragments into a fresh tube. Then incubate the pellet in a 15 milliliter tube with five milliliters of collagenase type one at 37 degrees Celsius under continuous agitation for 35 minutes. Separate the cells by resuspending with a one milliliter pipette and centrifuge at 150 times G for five minutes at four degrees Celsius.Clean the cells twice by resuspending the separated pellet in two milliliters of complete DMEM and centrifuging at 150 times G for five minutes at four degrees Celsius. For plating, resuspend the pellet in one milliliter of complete medium and add it to one well of a six well cell culture dish in a minimum amount of medium. Keep the plates undisturbed at 37 degrees Celsius with 5%carbon dioxide.After three days of incubation, confirm growth close to the tissue debris by observing the cells under the microscope. Once 1, 000 cells are visible, carefully remove the tissue debris with autoclaved tweezers and change the medium. Trypsinize the cells after reaching 70%confluency and transfer them to a 75 milliliter tissue culture dish.Before beginning the assay, clean the biosafety hood with 70%ethanol and warm the DMEM medium to 37 degrees Celsius. Next, seed 100, 000 cells per condition into six well plates in complete DMEM and culture at 37 degrees Celsius. After 24 hours, replace the supernatant medium with the calcifying medium and incubate the cells for seven days at 37 degrees Celsius, replacing the medium on the third day.After seven days, measure the calcium concentration in a 96 well plate by using the Arsenazo III reagent and recording the absorbance at 650 nanometers. In the representative analysis, various valve cell phenotypes were identified with immunofluorescent staining after three to five days of culture. Mouse VIC's expressed vimentin and alpha SMA, the significant markers of valve cells.Additionally, CD31 immunofluorescent staining verified no contamination from endothelial cells in VIC culture. Culturing VIC's with phosphate rich calcifying medium stimulate a calcification in cells, further confirmed with the red positive staining for calcium nodes. The protocol is based on a pool of three to five valve from different mice.The use of litter mate and three different biological replicates is important to validate the findings. The use of mouse valve cells is vital to understand the molecular pathway leading to aortic stenosis by isolating cells from transgenic mice. This protocol has been used previously to investigate the implication of pathway to receptor in mineral regression of calcified aortic valve.

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