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 mm HEPES. Prepare an aliquot of 50 mL of 1x phosphate-buffered saline (PBS). Keep the solutions on ice.</li>
	<li>Prepare 1 mg/mL and 4.5 mg/mL collagenase solutions, and use 5 mL of each solution in 15 mL tubes to perform the entire procedure. To prepare 5 mL of 1 mg/mL collagenase, mix 5 mg of collagenase with 2.5 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM, fetal bovine serum (FBS)-free) and 2.5 mL of 10 mM HEPES supplemented with antibiotics (1% penicillin-streptomycin from step 1.2). Filter the solutions through a 0.22 &#181;m filter to remove any contamination. 
	NOTE: Keep the solutions on ice to protect the enzymes.</li>
	<li>Warm the DMEM solution to 37 &#176;C before use in all the steps described below. Prepare complete medium by supplementing DMEM with 1% penicillin-streptomycin, 1% sodium pyruvate, 5 mL of 200 mM L-glutamine, 1 mL of mycoplasma elimination reagent (see the Table of Materials), and 10% FBS.</li>
</ol>
2. Isolation of valve cells
<ol>
	<li>To obtain 106 cells for the experiment, use five 8-week-old mice (minimum of three). Place the mouse in an induction chamber along with a small piece of tissue paper soaked with 1 mL of isoflurane, but do not allow contact with the tissue. To confirm that the animal is fully anesthetized; check for toe pinch reflex, and then euthanize the mouse by cervical dislocation. Use isoflurane to alleviate any pain prior to the cervical dislocation as the procedure described below is terminal.</li>
	<li>Place the mouse on a dissecting platform, and fix the paws with cannulas to hold it in place. Clean the chest and the abdomen with ethanol; open the abdomen and the chest with scissors. With small surgical scissors, cut between the left atrium and the left ventricle to exsanguinate the mouse. Perfuse the heart with 10 mL of cold 1x PBS to remove blood from the heart.</li>
	<li>Cut the heart, and keep 3 mm from the ascending aorta as shown in Figure 1B. Dissect the aortic valve under a stereomicroscope. Cut the heart horizontally in the middle of the ventricles (Figure 1C). Cut the left ventricle toward the aorta, and carefully dissect the aortic valve (Figure 1D-F). Pool the valves together in a small 35 mm tissue culture dish.</li>
	<li>Wash the isolated valves in a 75 mm cell culture dish with 5 mL of cold HEPES (10 mM) supplemented with antibiotics (1% penicillin-streptomycin) to remove blood (Figure 2). Prepare two 15 mL tubes of collagenase 1 mg/mL and 4.5 mg/mL as described above in step 1.3. 
	NOTE: After the dissection, manipulate the isolated valves in a sterile biosafety hood to minimize contamination.</li>
	<li>Incubate the valves in collagenase type I (1 mg/mL) for 30 min at 37 &#176;C with continuous shaking (Figure 2). Centrifuge the tube for 5 min at 150 &#215; g, wash the pellet once with 2 mL of HEPES (10 mM), and vortex for 30 s at high speed. Pour the contents of this tube into a 35 mm culture dish, and carefully transfer the fragments of tissue using thin tweezers into a new tube. 
	NOTE: At this stage, the VICs are still not dissociated from the tissue, and the pellet contains pieces of tissue. To avoid contamination with endothelial cells, do not centrifuge after vortexing in step 2.5.</li>
	<li>Incubate the pellet in a 15 mL tube with 5 mL of collagenase type I (4.5 mg/mL) at 37 &#176;C under continuous agitation for 35 min. Re-suspend the cells with a 1 mL pipette to separate the cells, and centrifuge at 150 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant, and re-suspend the pellet in 2 mL of complete DMEM. Centrifuge at 150 &#215; g for 5 min at 4 &#176;C. Repeat this step twice to clean the cells. 
	NOTE: The pellet will still have some tissue fragments.</li>
	<li>Re-suspend the pellet in 1 mL of complete medium, and plate the cells in one well of a 6-well cell culture dish in a minimum amount of medium to facilitate their attachment to the culture dish. Leave the cells, undisturbed, in a 37 &#176;C incubator with 5% carbon dioxide.</li>
	<li>After 3 days, check the cells under the microscope to verify good growth close to the tissue debris. Once 1,000 cells are visible under the microscope, carefully remove the tissue debris with autoclaved tweezers, and change the medium. 
	NOTE: The plate should not be disturbed; if the required number of cells are not observed, place the cell culture dish back in the incubator for another 2 days.</li>
	<li>When the cells are 70% confluent (2.5 &#215; 105), trypsinize and then transfer them to a 75 mm tissue culture dish.</li>
</ol>
3. Analysis of cell identity and morphology
NOTE: Immunofluorescence staining was used to study cell morphology and endothelial cell contamination.
<ol>
	<li>Clean the hood with 70% v/v/ ethanol. Place sterile coverslips (22 mm x 22 mm) in 6-well plates. 
	NOTE: To sterilize the coverslips, wash them with 70% ethanol, and keep them in the hood overnight under ultraviolet light.</li>
	<li>Seed 100,000 cells per well in a 6-well plate. After 24 h, wash the cells twice in 1x PBS, and fix them in 4% paraformaldehyde (PFA) for 20 min. Wash the cells again twice with 1x PBS. 
	NOTE: At this point, the cells could be kept in PBS at 4 &#176;C until the start of the staining procedure.</li>
	<li>To verify the purity of the VICs, use alpha-smooth muscle actin (&#945;SMA), vimentin, and cluster of differentiation 31 (CD31) to detect contamination with VECs.</li>
	<li>Prepare an aliquot of blocking buffer by mixing 500 &#181;L of normal serum (the same species as the secondary antibody), 9.5 mL of 1x PBS, and 30 &#181;L of Triton X-100. Incubate the cells in 2 mL of the blocking buffer for 1 h.</li>
	<li>Prepare the antibody dilution buffer containing 30 &#181;L of Triton X-100, 10 mL of 1x PBS, and 0.1 g of bovine serum albumin (BSA).</li>
	<li>Take an empty tips box, fill half of the box with water to create a humid chamber. Cover the tip holder with a wet tissue and then with a sheet of parafilm.</li>
	<li>Take 1 &#181;L of the primary antibody, and mix it with 100 &#181;L of the dilution buffer prepared in step 3.5. Place 50 &#181;L of the diluted antibody on the parafilm. Take the coverslips from the wells, flip them over, and place them on the top of the drops of antibody; incubate the cells overnight with the antibody.</li>
	<li>Add 1 mL of PBS in the 6-well plate. Carefully take out the coverslip from the parafilm, flip it over, and place it in the well. Wash the cells with a continuous gentle agitation for 5 min. Replace the PBS with fresh PBS; wash the cells 3 times.</li>
	<li>Incubate the cells with the diluted secondary antibody (1/500) (Alexa-488, Alexa-555) for 1 h. Add 1 &#181;L of the secondary antibody to 500 &#181;L of the antibody dilution buffer (prepared in step 3.5). Cover the plate with aluminum foil. Wash the cells 3 times with 1 mL of 1x PBS with continuous agitation.</li>
	<li>Mount the coverslips with 50 &#181;L of 4&#8242;,6-diamidino-2-phenylindole (DAPI)-mounting medium, and observe the cells under the microscope to analyze the morphology of cells and VEC contamination.</li>
</ol>
4. In vitro calcification assay
<ol>
	<li>Clean the hood with 70% ethanol, warm the DMEM medium to 37 &#176;C.</li>
	<li>Seed 100,000 cells/condition into 6-well plates in complete DMEM, and culture for 24 h at 37 &#176;C.</li>
	<li>Prepare the calcifying medium by mixing 2 mM of NaH2PO4, 10-7 M insulin, and 50 &#181;g/mL ascorbic acid in DMEM with 5% FBS. For 93 mL of DMEM, add 5 mL of FBS, 1 mL of antibiotics (final concentration 1%), 1 mL of sodium pyruvate (100 mM), 27.5 mg of NaH2PO4, 5.8 &#181;L of insulin, and 5 mg of ascorbic acid. 
	NOTE: Filter the solution using a 0.22 &#181;m filter before use.</li>
	<li>After 24 h, replace the supernatant medium with the calcifying medium. Incubate the cells for 7 days at 37 &#176;C. On the 3rd day, replace with fresh calcifying medium, and place the plate back in the incubator to complete the 7 days of treatment.</li>
	<li>After 7 days, remove the medium, and wash the cells twice with 2 mL of 1x PBS. Incubate the cells in 1 mL of 0.6 N hydrochloric acid (HCl) for 24 h at 37 &#176;C. Collect the HCl in a 1.5 mL tube, and evaporate it in a rotary evaporator. Re-suspend the contents of all the tubes in 60 &#181;L of HCl. 
	NOTE: The drying procedure is important to concentrate the solution and to have the same volume for each condition.</li>
	<li>Use a 96-well plate to measure calcium concentration by using Arsenazo III reagent, available in a ready-to-use kit (see the Table of Materials for more details).</li>
	<li>Prepare a calcium standard solution of 10 mg/dL concentration. Weigh 10 mg of calcium hydroxide (Ca(OH)2) and dissolve in 100 mL of distilled water.</li>
	<li>In a clear 96 well plate, pipet 2 &#181;L of blank solution (HCl, 0.6 N), the standard solution, the sample per well (10 mg/dL), and the samples. Perform the experiment in triplicate to verify the pipetting variability. Add 200 &#181;L of the reagent for each condition. 
	NOTE: Samples above 15 mg/dL should be diluted 1:1 with saline, re-assayed, and the result multiplied by two.</li>
	<li>Incubate the reaction for 15 min at room temperature. 
	NOTE: The reaction is stable for 60 min.</li>
	<li>Read and record the absorbance of the plate at 650 nm. Use the following formula to calculate the amount of calcium in the samples: 
	Calcium (mg/mL) = (Absorbance of sample/absorbance of standard) &#215; Concentration of standard</li>
</ol>

<ol>
	<li>Rostagno, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+disease+in+elderly.">Heart valve disease in elderly.</a> World Journal of Cardiology. 11 (2), 71-83 (2019).</li><li>Stewart, B. F., 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=Clinical+factors+associated+with+calcific+aortic+valve+disease.+Cardiovascular+Health+Study.">Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study.</a> Journal of the American College of Cardiology. 29 (3), 630-634 (1997).</li><li>Marquis-Gravel, G., Redfors, B., Leon, M. B., G&#233;n&#233;reux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+treatment+of+aortic+stenosis.">Medical treatment of aortic stenosis.</a> Circulation. 134 (22), 1766-1784 (2016).</li><li>Spitzer, E., 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=Aortic+stenosis+and+heart+failure:+disease+ascertainment+and+statistical+considerations+for+clinical+trials.">Aortic stenosis and heart failure: disease ascertainment and statistical considerations for clinical trials.</a> Cardiac Failure Review. 5 (2), 99-105 (2019).</li><li>Hinton, R. B., Yutzey, K. 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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 sources of possible contagion to protect the cells from bacterial or mycoplasma contamination. Indeed, it is crucial to autoclave all the surgical tools prior to starting the experiments. The HEPES solution should be supplemented with 1% antibiotics to minimize bacterial infection. Furthermore, mycoplasma may cause cytopathology and consequently interfere with every parameter measured in cell culture10.
Plating cells in small culture dishes with lower volume of culture medium is critical for VIC growth and proliferation. Letting the tissue settle and adhere to the cell culture dish permits cell migration from the tissue to the dish wall. Given that isolated cells from young mice proliferate faster, it is recommended to transfer cells to a larger culture dish of 75 cm2 after 5 days of culture. Maintaining cells to 80% confluence is crucial to minimize the differentiation of VICs to a myofibroblast phenotype8.
As shown by immunofluorescence imaging, the isolated valve cells show a fibroblast-like phenotype. VICs have an elongated cytoplasm and express both vimentin and &#945;SMA as described by previous studies. The present work confirmed that the mouse VIC phenotype is similar to that previously described for porcine VICs11 and human VICs12. Most in vitro studies on aortic stenosis are performed on cells from large animals8,11. The key disadvantage of porcine VICs is their spontaneous differentiation to an osteoblast phenotype in vitro even in normal media13. However, mouse VICs do not calcify spontaneously even at higher passages.
Mouse VICs differentiate to the osteoblast phenotype in response to calcifying medium using ascorbic acid, insulin, and phosphate stimulation. This article describes a quantitative method of calcium measurement using a kit and a qualitative method using Alizarin red staining. Both methods showed significant increase of calcification in response to calcifying medium treatment. The calcium measurement kit is the gold standard method, which offers an exact quantitative calcium measurement14.
In the Arsenazo III reagent, magnesium interference is prevented by the inclusion of 8-hydroxyquinoline sulfonate. Calcium reacts with the reagent to form a purple-colored complex, which absorbs at 650 nm. The intensity of the color is proportional to the calcium concentration. The accuracy of the Arsenazo-III reagent was previously validated with atomic absorption spectrophotometry. The same method is used in clinical laboratories to measure total calcium concentration in biological fluids14. The calcification in aortic stenosis is mainly hydroxyapatite, as shown with dispersive x-ray energy scanning electron microscopy analysis7,12,15. Indeed, it is important to analyze the calcification of the cell membrane rather than free calcium to more accurately mimic the calcification of the aortic valve tissue.
Mice represent a good source of VICs for the study of molecular mechanisms leading to aortic valve calcification. However, keep in mind that VICs in vitro are not similar to VICs in living valves. Another limitation is the fact that a pool of valves from 3-5 mice is needed to make a single cell culture. The pool should be from littermate mice to minimize variations. In addition, experiments should be performed in triplicate to confirm all findings. However, the use of the entire aortic valve in the culture can alleviate this limitation. Nevertheless, these in vitro studies must be validated in human tissue to strengthen the findings.

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><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&amp;nbsp; (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>A2916801</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>&amp;nbsp;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 endothelial cells contamination (Figure 3D). In addition, mouse VICs express vimentin and &#945;-SMA, which are the major markers of valve cells (Figure 3B,C).
Cell mineralization in vitro 
A calcium reagent kit was used to measure the calcium concentration; cells treated with calcifying medium have higher calcium concentration compared to non-treated cells (Figure 4A). The concentration of calcium was normalized with the total protein concentration. Alizarin red staining confirmed the calcium-reagent kit measurements by showing red positive calcium nodes (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62419/62419fig01.jpg" /> 
Figure 1: Description of valve dissection. (A)&#160;Representative image of all the surgical instruments needed for the dissection, scissors 2 is needed to open the skin of the mouse and scissors 3 to open the chest. Tweezers 5 and 6 are needed to hold the skin and open the chest. (B) Leave 3 mm of tissue from the aorta (black arrow). (C) Cut the heart in the middle of the ventricles with scissors number 4. (D) Open the heart toward the aortic valve with scissors 3. Use the thin tweezers 7 and 8 to carefully dissect the aortic valve. The valve is visible and has some black dots that are characteristic of mice valve tissue (blue arrow). (E) Increase the magnification to better visualize the aortic valve. Isolate the valve with the small scissors 4; (F) maintain the tissue with tweezers 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62419/62419fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62419/62419fig02.jpg" /> 
Figure 2: Representative description of mouse valve cell isolation. Abbreviations: HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RT = room temperature; DMEM = Dulbecco&#39;s modified Eagle medium; FBS = fetal bovine serum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62419/62419fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62419/62419fig03.jpg" /> 
Figure 3: Mouse valve cell phenotype. Microscopic view of (A) freshly isolated valve cells. Immunofluorescence staining showing (B) vimentin-positive cells and (C) &#945;-SMA. Cells are negative for (D) CD31 staining. Scale bars = 200 &#181;m. Abbreviations: DAPI = 4&#8242;,6-diamidino-2-phenylindole; CD31 = cluster of differentiation 31; &#945;-SMA = alpha-smooth muscle actin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62419/62419fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62419/62419fig04.jpg" /> 
Figure 4: In vitro calcification assay. (A) Phosphate-rich calcifying medium induced VIC calcification in vitro, which was measured with a reagent kit. (B) Microscopic image showing red positive staining (right) for calcium nodes. (C) Alizarin red staining showed positive calcium nodes (black arrow) of VICs in response to calcifying medium. Scale bars = 100 &#181;m. Abbreviations: CTL- = Control; mVICs = mouse valvular interstitial cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62419/62419fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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) ...

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3.9K Views.  The Icahn School of Medicine at Mount Sinai. 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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Developmental Biology

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Bioengineering

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 ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Medicine

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

Immunology and Infection

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

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.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

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.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Isolation of Mouse Valve Interstitial Cells: An Enzymatic Method to Isolate and Culture VICs from Mouse Aortic Valve

Source: Bouchareb, R. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/62419" target="_blank">Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve&#160;In Vitro</a>.&#160;J. Vis. Exp. (2021)
In this video, we demonstrate the isolation of aortic valve interstitial cells from the heart of an adult mouse using a two-step collagenase procedure and their subsequent culture in low volume of growth media.

Source: Bouchareb, R. et al. Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve&#160;In Vitro.&#160;J. Vis. Exp. ...

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Adult

Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

Source: Bouchareb, R. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62419">Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.</a>&#160; J. Vis. Exp. &#160;(2021).
This video demonstrates an assay technique for assessing the calcification of the aortic valve interstitial cells in vitro. The assay helps to quantify the calcium concentration using the arsenazo III reagent, a calcium-sensitive dye that reacts with calcium ions to form a purple-colored calcium-arsenazo complex.

Source: Bouchareb, R. et al., Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.&#160; J. Vis. Exp. ...

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

Summary

Explore More Videos

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Isolation of Valvular Endothelial Cells

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Isolation of Murine Valve Endothelial Cells

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Isolation of Mouse Valve Interstitial Cells: An Enzymatic Method to Isolate and Culture VICs from Mouse Aortic Valve

Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells