We would like to thank Jason Dobbins for insightful discussion and critical reading of this manuscript. We would like to acknowledge the Center for Organ Recovery and Education for their help and support and thank tissue donors and their families for making this study possible. All patient samples are collected from individuals enrolled in studies approved by the institutional review board of the University of Pittsburgh in accordance with the Declaration of Helsinki. Cadaveric tissues obtained via the Center for Organ Recovery and Education (CORE) were approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents (CORID).
Some figures created with Biorender.com.
CSH is supported by the National Heart, Lung, and Blood Institute K22 HL117917 and R01 HL142932, the American Heart Association 20IPA35260111.

All patient samples are collected from individuals enrolled in studies approved by the institutional review board of the University of Pittsburgh in accordance with the Declaration of Helsinki. Cadaveric tissues obtained via the Center for Organ Recovery and Education (CORE) were approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents (CORID).
1. Approval and safety
<ol>
	<li>Obtain Institutional Review Board (IRB) approval or an exempt memo for any collection of patient samples or cadaveric tissues in accordance with the Declaration of Helsinki.</li>
	<li>Take required institutional training to work with human tissues such as Bloodborne Pathogens Training, Biomedical Human Subjects Research, Privacy and Information Security, and Transportation and Shipping of Biological Materials.</li>
</ol>
2. Logistics and preparation
<ol>
	<li>Surgical Samples
	<ol>
		<li>Ensure that a refrigerator is available and located near the operating room. Keep 50 mL conical tubes pre-labeled with de-identified nomenclature containing 40 mL of sterile aliquots of cold storage solution in this fridge for use by surgical staff. These tubes are stable at 4 &#176;C until expiration date on the original package.</li>
		<li>Upon extraction, place valve tissue in these tubes. Place the tubes in a sealed secondary container such as a leak-proof plastic bag or a plastic container that is labeled with a biohazard label. Tissues are picked up and transported on ice according to institutional protocols for transporting biohazards.</li>
	</ol>
	</li>
	<li>Cadaveric samples
	<ol>
		<li>Submerge recovered organs in cold storage solution, place in a sealed secondary containment vessel, and transport on ice according to institutional protocols for transporting biohazards.</li>
	</ol>
	</li>
</ol>
3. Reagents preparation
<ol>
	<li>Make collagen-coated plates at least the day before.
	<ol>
		<li>In a 50 mL conical tube, mix 5 mL of isopropyl alcohol, 8.7 mL of acetic acid, and 0.5 &#181;g of collagen I powder. Bring up to 50 mL with sterile water. Mix and filter through a 0.45 &#181;m filter.</li>
		<li>Under a sterile cell culture hood, add enough collagen solution to 6 well plates and 10 cm dishes to just cover the entire bottom. Cover the plate and let sit for 4-6 h at room temperature. Remove the excess solution with a sterile pipette, place in a new sterile 50 mL conical tube, and save at 4 &#176;C to make additional plates. Solution can be stored at 4 &#176;C for several months.</li>
		<li>Dry the plates in a 37 &#176;C incubator overnight, and subsequently store them in a resealable bag at 4 &#176;C. Collagen-coated plates and dishes can be stored for several months.</li>
	</ol>
	</li>
	<li>Autoclave the following items: tissue forceps, tissue scissors, cotton swabs and gauze pads.</li>
	<li>VEC growth media: Prepare and use endothelial cell growth medium according to manufacturer&#39;s protocols. Store in the dark at 4 &#176;C. Warm to 37 &#176;C just before use on cells, do not leave media in warming bath longer than necessary (10-15 min is sufficient). Use the media within one month of preparation.</li>
	<li>VIC growth media: Supplement DMEM base medium (4.5 g/L glucose, L-glutamine supplement, 110 mg/L sodium pyruvate)30 with 10% heat-inactivated FBS and 100 I.U./mL penicillin and 100 &#956;g/mL streptomycin. Store in the dark at 4 &#176;C for up to 3 months. Warm to 37 &#176;C just before use on cells, do not leave media in warming bath longer than necessary (10-15 min is sufficient).</li>
	<li>Make sterile rinsing solution just before use. Supplement sterile PBS with 2.5 &#181;g/mL fungicide, 0.05 mg/mL gentamicin and 5 &#181;g/mL bactericide.</li>
	<li>Make sterile collagenase solution just before use. Add 5 mg of collagenase II to 5 mL of sterile fresh DMEM base medium. Mix well and sterilize the solution by passing through a 0.45 &#181;m filter. Keep on ice until use.</li>
</ol>
4. Tissue preparation and processing
NOTE: Institutional approval for use of human tissues must be obtained prior to beginning work. While handling tissues, the following personal protective equipment (PPE) must be worn: a disposable liquid barrier wrap-around gown, or a dedicated button front lab coat with a liquid-barrier wrap around apron and disposable sleeve clovers; a full face shield, or safety glasses with a surgical mask; double gloves; close-toed shoes; and clothing to cover the legs. Comprehensive workflow diagrams of the tissue preparation for calcification assessment (Section 5) and cell isolation (Sections 6 and 7) are illustrated in Figure 1A,B respectively.
<ol>
	<li>Upon receipt of cadaveric organ specimen, excise the aortic root and submerge in a 50 mL conical tube of sterile rinsing solution. Upon receipt of surgical specimen, remove from transport vessels and submerge in a 50 mL conical tube of sterile rinsing solution. Place tube containing the tissue in ice bucket on rocker and mix for 10 min at room temperature (RT). 
	NOTE: Processing tissues as close as possible to the time of extraction will yield the best cell recovery, however viable cells can be collected upwards of 61 h post excision, and data show that VICs are more robust than VECs as time increases. If tissue cannot be processed immediately, when possible, remove tissue, perform step 4.1, and then put the tissue back in fresh sterile cold storage solution and keep at 4 &#176;C until ready to proceed. Valves collected during night or weekend surgeries can be stored in 40 mL cold storage solution at 4 &#176;C and still are able to yield viable cells more than 2 days post extraction.</li>
	<li>Spray down tubes with 70% ethanol and move to a sterile hood. Remove tissue and excise two valve leaflets (Figure 2A). Place one leaflet in a cryogenic vial (or 2-3 vials if several smaller pieces are needed for future analysis) and snap freeze by dropping in liquid nitrogen then store at -80 &#176;C.
	<ol>
		<li>If the valve is bicuspid, excise only one leaflet. Using scissors, cut the leaflet in half from the nodule to the hinge. Use one half of the leaflet for snap freezing and the other half for step 4.3.</li>
	</ol>
	</li>
	<li>Process the second leaflet for paraffin embedding to assess calcification content by cutting it in half from nodule to hinge. Place both pieces in a cassette that is submerged in 4% paraformaldehyde (PFA), then place on a rocker at RT for a minimum of 2 h but no more than 4 h. 
	NOTE: Longer fixation times create more background with immunofluorescent staining. Once steps 4.2 and 4.3 are performed, move immediately to Section 6 and come back to step 4.4 after step 6.12.</li>
	<li>After fixation, wash the tissues by submerging in fresh PBS for 1 h 3-4 times. After these washes, samples can stay in PBS at 4 &#176;C for several months if needed. Proceed to next step just before embedding.</li>
	<li>Gradually change from PBS to 70% ethanol. Wash 30-60 min each step with 1:4 70% ethanol:PBS; 1:1 70% ethanol:PBS, 4:1 70% ethanol:PBS; 70% ethanol.</li>
	<li>Embed the tissue such that sections will reveal the three layers of the leaflet (Figure 1A) according to established protocols31. Alternatively, and if available, bring the tissue to a pathology core for embedding and cutting.</li>
	<li>After handling tissues, dispose or store PPE as appropriate and wash hands immediately. Decontaminate all equipment, surfaces, and solid and liquid wastes with a 1:10 dilution of bleach or detergent disinfectant. Allow 20 min for decontamination, then follow with a 70% ethanol rinse. Treat the cell culture hood with mycoplasma spray according to manufacturer&#39;s instructions.</li>
</ol>
5. Von Kossa staining for calcium content
NOTE: This can be done well after cell isolation and line establishment but be sure to link the calcification level of the tissue to documents pertaining to the primary cell line established.
<ol>
	<li>Cut 5 or 10 &#181;m thick paraffin slices onto glass slides and bake slides at 65 &#176;C for 1 h, then cool to RT.</li>
	<li>Using fresh solutions, deparaffinize slides by submerging as following: 100% xylene for 30 min, 2x; 100% ethanol for 3 min, 2x;&#160;90% ethanol for 3 min; 80% ethanol for 3 min; 70% ethanol for 3 min; 50% ethanol for 3 min; ultrapure water for 3 min; keep in ultrapure water until next steps. 
	NOTE: Times above are minimum, each step may go longer.</li>
	<li>Proceed with Von Kossa staining according to manufacturer&#39;s protocols.</li>
	<li>Assess full valve area microscopically and assign a control tissue (no sign of calcification) or CAVD (any evidence of calcification, Figure 2B).</li>
</ol>
6. Valve Endothelial Cell (VEC) isolation, expansion, and confirmation
<ol>
	<li>In a sterile cell culture hood, open the conical tube containing the remaining valve leaflet and place the leaflet in a new 50 mL conical tube filled with ice cold PBS. Cap tube and gently invert or place on a rocker for 2 min at RT.</li>
	<li>Remove tissue to a 60 mm dish filled with 5-7 mL of cold collagenase solution. Using forceps, dip both sides of the leaflet in the solution 3-4 times. Incubate the tissue for 5-10 min in the cell culture incubator at 37 &#176;C, rocking the tissue gently every 2 min 3-4 times.</li>
	<li>Remove 2 mL of the solution from the dish and place in a sterile 15 mL conical tube. Place forceps at the nodule and use a dry sterile cotton swab to swipe from the forceps to the hinge, twirling the swab while moving it along the leaflet. Between each swipe, swish the swab in the solution in the 15 mL conical tube to remove the cells. Repeat to fully swab the surface of the tissue, then flip over and repeat on the other side.</li>
	<li>Holding the valve leaflet with forceps in one hand, and a 1 mL pipette in the other, rinse the surfaces of the leaflet with the solution in the dish. Once rinsed, transfer all the solution containing the VECs in the dish into the same 15 mL conical tube with the cells from the swab and proceed to Step 6.5. Place the remaining valve tissue in a new 15 mL conical tube with 7 mL of sterile collagenase solution and proceed to Step 7.1.</li>
	<li>Centrifuge the tube containing the VECs at 180 x g for 5 min to pellet the isolated VECs. Aspirate the supernatant and resuspend in 3 mL of VEC growth media. Centrifuge one more time and remove the supernatant. Resuspend the cells in 1 mL of growth media and determine the number of cells using a hemocytometer and trypan blue.</li>
	<li>Resuspend VEC pellet in 2 mL of VEC growth media and plate the cells in a collagen pre-coated well of a 6 well plate at approximately 5 x 105 cells/cm2. Let the cells grow at least 3-4 days, then remove media and replenish with fresh media. Repeat media changing every 4 days. VECs will grow in cobblestone shaped patches (Figure 3B, left panels).</li>
	<li>When VEC patches cover &#62;80% of the plate, split cells at about 1.3 x 104 cells/cm2 depending on how fast they grow (if they reach 80% in less than 1 week split at a slightly lower number of cells/cm2).</li>
	<li>To split, wash cells two times with 2 mL DPBS then add enough dissociation reagent to just cover the surface of the cells. Incubate for 2-3 min at 37 &#176;C, checking to make sure cells are not over incubated. Stop digestion by adding equal volume of VEC growth media and transfer liquid to a 15 mL tube. Centrifuge at 180 x g for 5 min, remove supernatant, and resuspend in appropriate volume of media for number of wells/plates needed. 
	NOTE: Once expanded into a 10 cm plate VECs may sometimes lose their morphology and change phenotype as they proliferate. This tends occurs when VECs are seeded at a low confluency during the establishment and expansion of the cell line.</li>
	<li>To guarantee a pure culture consider utilizing CD31+ superparamagnetic beads with every splitting.</li>
	<li>For 1 x 108 or fewer cells (one 10 cm dish or less), prepare 25 &#181;L of superparamagnetic beads by washing according to manufacturer&#39;s protocols. 
	NOTE: Step 6.10 is recommended to be performed before trypsinization of VECs.</li>
	<li>Detach cells as in step 6.8 but resuspend in 500 &#181;L of PBS with 0.1% BSA, pH 7.4, and place in a 2 mL centrifuge tube. Add 500 &#181;L of washed and resuspended beads to the 2 mL tube of cells and incubate on a rotator for 20 min at 4 &#176;C.</li>
	<li>Place tubes on the magnet for 2 min. While tube is still in the magnet, carefully remove supernatant. Remove tube from magnet, add 1 mL fresh PBS with 0.1% BSA, pipette gently 2-3 times, then place back on magnet for 2 min. Repeat 2 more times. After final removal of buffer, resuspend cells in growth media in volume needed for replating. 
	NOTE: Cells will still have beads, but these will not affect growth and will be removed in subsequent passages.</li>
	<li>Once cells have been expanded, in addition to morphology, confirm VEC phenotype by positive staining for immunofluorescent markers such as von Willebrand factor (vWF), cadherin 5 (CDH5), or PECAM-1/CD31, and negative staining for&#160;VIC markers such as calponin 1 (CNN) or alpha-2 smooth muscle actin (&#945;SMA, Figure 4, left panels).</li>
</ol>
7. Valve Interstitial Cell (VIC) isolation, expansion, and storage
<ol>
	<li>As stated in step 6.4, after removing VECs from the valve tissue, the leaflet is&#160;placed in a 15 mL conical tube with 7 mL collagenase solution. Incubate for 12 h in the cell culture incubator with the cap slightly open to allow gas exchange. Successful isolation of VICs can still occur with up to 18 h in collagenase solution.</li>
	<li>After incubation, in a sterile cell culture hood, mix the tissue gently by pipetting with a serological pipette to ensure the release of VICs from the leaflet tissue.</li>
	<li>Remove cell suspension and pass through a 0.70 &#956;m filter into a 50 mL conical tube.</li>
	<li>Add 7 mL of VIC growth medium to the 50 mL tube and centrifuge at 180 x g for 5 min. Aspirate supernatant and resuspend cell pellet in 1 mL of VIC growth media and determine the cell number. Plate the VICs in a 60 mm tissue-culture treated dish at 1.3 x 104 cells/cm2.</li>
	<li>Let the cells grow at least 1-2 days before replacing media. Remove media and wash twice DPBS to remove residual debris and replace with fresh growth medium. Replenish medium every 2-3 days. VICs will grow in fibroblast shape (Figure 3B, right panels).</li>
	<li>When VICs reach a confluency of &#62;90%, wash twice with DPBS to remove the excess media and detach the cells by adding appropriate volume of pre-warmed dissociation reagent to cover the plate (i.e., 2-3 mL per 10 cm dish). Incubate the dish in a 37 &#176;C incubator. VICs will detach from the dish after 2-3 min of incubation. Add 4-6 mL of pre-warmed media. 
	NOTE: If cells take longer than 3 min to lift off the plate, the dissociation reagent may have lost potency. If needed, a cell scraper may be used to gently lift the cells.</li>
	<li>Transfer the cell suspension to a tube and gently centrifuge at 180 x g for 5 min. After removing the supernatant, gently resuspend the cell pellet in pre-warmed VIC growth media and determine the number of viable cells by using a hemocytometer and trypan blue. Seed the viable cells at a 1:2 density of the original dish (i.e., ~ 1 &#215; 106 cells per 10 cm dish or 1.3 x 104 cells/cm2).</li>
	<li>Assess VIC phenotype by positive staining for immunofluorescent markers such as &#945;SMA, CNN, or SM22&#945; and negative staining for VEC markers such as CD31, CDH5, or vWF (Figure 4, right panels). 
	NOTE: The protocol workflow presented here unbiasedly selects one leaflet for VECs and VICs isolation, while the remaining leaflets are utilized for Von Kossa staining (Section 5) and snap freezing.</li>
</ol>
8. Long-term cell storage
<ol>
	<li>Once expanded, freeze cells down for long-term storage. Wash cells twice with 2-5 mL DPBS then add enough dissociation reagent to detach cells as in steps 6.8 and 7.6. Stop digestion by adding equal volume of VEC or VIC growth media and transfer cell suspension to a 15 mL tube.</li>
	<li>Determine the number of viable cells by using a hemocytometer and trypan blue.</li>
	<li>Centrifuge at 180 x g for 5 min.</li>
	<li>Resuspend cells in chilled conditioned growth media to a cell density of ~ 3 &#215; 106 cells/mL.</li>
	<li>Gently with swirling, add an equal volume of chilled 2x cryopreservation medium. This will bring cell concentration to ~ 1.5 &#215; 106 cells/mL.</li>
	<li>Aliquot 1 mL into cryopreservation vials. Place vials in a cell freezing container, close, and invert 5-6 times to ensure cells remain suspended. Place container at -80 &#176;C for 6-72 h or according to freezing container protocol. Remove vials from -80 &#176;C and transfer to liquid nitrogen for long term storage.</li>
</ol>

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<li>Jian, B., Narula, N., Li, Q. Y., Mohler, E. R., Levy, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progression+of+aortic+valve+stenosis%3A+TGF-beta1+is+present+in+calcified+aortic+valve+cusps+and+promotes+aortic+valve+interstitial+cell+calcification+via+apoptosis%5BTitle%5D)+AND+%22Annals+of+Thoracic+Surgery%22%5BJournal%5D)">Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis.</a> Annals of Thoracic Surgery. 75, 465(2003).</li>
<li>Bogdanova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interstitial+cells+in+calcified+aortic+valves+have+reduced+differentiation+potential+and+stem+cell-like+properties%5BTitle%5D)+AND+%22Science+Reports%22%5BJournal%5D)">Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties.</a> Science Reports. 9, 12934(2019).</li>
<li>Gendron, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+aortic+valve+interstitial+cells+display+proangiogenic+properties+during+calcific+aortic+valve+disease%5BTitle%5D)+AND+%22Arteriosclerosis%2C+Thrombosis%2C+and+Vascular+Biology%22%5BJournal%5D)">Human aortic valve interstitial cells display proangiogenic properties during calcific aortic valve disease.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (1), 415-429 (2021).</li>
<li>Wirrig, E. E., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conserved+transcriptional+regulatory+mechanisms+in+aortic+valve+development+and+disease%5BTitle%5D)+AND+%22Arteriosclerosis%2C+Thrombosis+and+Vascular+Biology%22%5BJournal%5D)">Conserved transcriptional regulatory mechanisms in aortic valve development and disease.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 34, 737-741 (2014).</li>
<li>Honda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mouse+model+of+aortic+valve+stenosis+induced+by+direct+wire+injury%5BTitle%5D)+AND+%22Arteriosclerosis%2C+Thrombosis+and+Vascular+Biology%22%5BJournal%5D)">A novel mouse model of aortic valve stenosis induced by direct wire injury.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 34, 270-278 (2014).</li>
<li>Sider, K. L., Blaser, M. C., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+calcific+aortic+valve+disease%5BTitle%5D)+AND+%22International+Journal+of+Inflammation%22%5BJournal%5D)">Animal models of calcific aortic valve disease.</a> International Journal of Inflammation. 2011, 364310(2011).</li>
<li>Gould, R. A., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+valvular+endothelial+cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isolation of valvular endothelial cells.</a> Journal of Visualized Experiments. (46), e2158(2010).</li>
<li>Miller, L. J., Lincoln, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+murine+valve+endothelial+cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isolation of murine valve endothelial cells.</a> Journal of Visualized Experiments. (90), e51860(2014).</li>
<li>Selig, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+hyperinsulinemia+and+hyperglycemia+on+valvular+interstitial+cells+-+A+link+between+aortic+heart+valve+degeneration+and+type+2+diabetes%5BTitle%5D)+AND+%22Biochimica+Biophysica+Acta+Molecular+Basis+of+Disease%22%5BJournal%5D)">Impact of hyperinsulinemia and hyperglycemia on valvular interstitial cells - A link between aortic heart valve degeneration and type 2 diabetes.</a> Biochimica Biophysica Acta Molecular Basis of Disease. 1865, 2526-2537 (2019).</li>
<li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraffin+embedding+tissue+samples+for+sectioning%5BTitle%5D)+AND+%22CSH+Protocols%22%5BJournal%5D)">Paraffin embedding tissue samples for sectioning.</a> CSH Protocols. 2008, (2008).</li>
<li>Martinsson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+trends+in+the+incidence+and+prognosis+of+aortic+stenosis%3A+A+nationwide+study+of+the+Swedish+population%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Temporal trends in the incidence and prognosis of aortic stenosis: A nationwide study of the Swedish population.</a> Circulation. 131, 988-994 (2015).</li>
<li>Rabkin-Aikawa, E., Farber, M., Aikawa, M., Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+and+reversible+changes+of+interstitial+cell+phenotype+during+remodeling+of+cardiac+valves%5BTitle%5D)+AND+%22Journal+of+Heart+Valve+Diseases%22%5BJournal%5D)">Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.</a> Journal of Heart Valve Diseases. 13, 841-847 (2004).</li>
<li>St Hilaire, C., Carroll, S. H., Chen, H., Ravid, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+induction+of+adenosine+receptor+genes+and+its+functional+significance%5BTitle%5D)+AND+%22Journal+of+Cell+Physiology%22%5BJournal%5D)">Mechanisms of induction of adenosine receptor genes and its functional significance.</a> Journal of Cell Physiology. 218, 35-44 (2009).</li>
<li>Xu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-type+transcriptome+atlas+of+human+aortic+valves+reveal+cell+heterogeneity+and+endothelial+to+mesenchymal+transition+involved+in+calcific+aortic+valve+disease%5BTitle%5D)+AND+%22Arteriosclerosis%2C+Thrombosis+and+Vascular+Biology%22%5BJournal%5D)">Cell-type transcriptome atlas of human aortic valves reveal cell heterogeneity and endothelial to mesenchymal transition involved in calcific aortic valve disease.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 40, 2910-2921 (2020).</li>
</ol>

IS receives institutional research support from Atricure and Medtronic and serves as a consultant for Medtronic Vascular. None of these conflicts are related to this work. All other Authors have nothing to disclose.

Obtaining control and disease tissues from humans is critical for in vitro and ex vivo disease modeling; however, while one often speaks about the challenges of bridging the gap between bench to bedside, the reverse order - going from the surgical suite to the bench - is often just as daunting a gap. Essential for a basic scientist to obtain primary human tissue specimens is a collaboration with an invested surgeon scientist who has a team of nurses, surgical technicians, physician assistants, medical students and reside...

Calcific aortic valve disease (CAVD) is a chronic pathology characterized by inflammation, fibrosis, and macrocalcification of aortic valve leaflets. Progressive remodeling and calcification of the leaflets (termed aortic sclerosis) can lead to the obstruction of blood flow (aortic stenosis) which contributes to stroke and leads to heart failure. Currently the only treatment for CAVD is surgical or transcatheter aortic valve replacement (SAVR and TAVR, respectively). There is no non-surgical option to halt or reverse CAVD progression, and without valve replacement, mortality rates approach 50% within 2-3 years1,2,3. Defining the underlying mechanisms driving this pathology will identify potential novel therapeutic approaches.
In a healthy adult, aortic valve leaflets are approximately one millimeter thick, and their main function is to maintain the unidirectional flow of blood out of the left ventricle4. Each of the three leaflets is comprised of a layer of valve endothelial cells (VECs) that lines the outer surface of the leaflet and functions as a barrier. VECs maintain valve homeostasis by regulating permeability, inflammatory cell adhesion, and paracrine signaling5,6,7. Valve interstitial cells (VICs) comprise the majority of cells within the valve leaflet8. VICs are arranged in three distinctive layers in the leaflet. These layers are known as the ventricularis, the spongiosa, and the fibrosa9. The ventricularis faces the left ventricle and contains collagen and elastin fibers. The middle layer, the spongiosa, contains high proteoglycan content that provides shear flexibility during the cardiac cycle. The outer fibrosa layer is located close to the outflow surface on the aortic side and is rich in Type I and Type III fibrillar collagen which provide strength to maintain coaptation during diastole10,11,12. VICs reside in a quiescent state, however, factors such as inflammation, remodeling of the extracellular matrix (ECM), and mechanical stress may disrupt VIC homeostasis8,9,13,14,15,16. With loss of homeostasis, VICs activate and acquire a myofibroblast-like phenotype capable of proliferation, contraction, and secretion of proteins that remodel the extracellular millieu17. Activated VICs can transition into calcifying cells which is reminiscent of the differentiation of a mesenchymal stem cell (MSC) into an osteoblast15,17,18,19,20,21,22,23,24,25.
Calcification appears to initiate in the collagen-rich fibrosa layer from contributions of both VECs and VICs but expands and invades the other layers of the leaflet8. Thus, it is clear that both VECs and VICs respond to stimuli to upregulate the expression of osteogenic genes, however, the precise events driving the activation of osteogenic genes, as well as the complex interplay between the cells and the extracellular matrix of the leaflet, remain ill-defined. Murine models are not an ideal source to study non-genetic drivers of CAVD pathogenesis, as mice do not develop CAVD de novo26,27, hence the use of primary human tissues and the primary cell lines isolated from these tissues is necessary. In particular, obtaining these cells in high numbers and good quality is imperative, as the field of 3D cell cultures and organoid modeling is expanding and is likely to become an ex vivo human-based alternative to murine models.
The purpose of the present method is to share a workflow that has established the conditions to efficiently isolate and grow VECs and VICs obtained from surgically removed valves from human donors. Previous studies have shown&#160;successful isolation of VECs and VICs from porcine28&#160;and murine valves29, to our knowledge this is the first to describe the isolation of these cells in human tissues. The protocol described here is applicable to human excised valves and greatly circumvents and improves the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing by introducing the utilization of a cold storage solution, a buffered solution clinically utilized in organ transplants that greatly stabilizes cells of the excised tissue. The protocol described here also shows how to determine cell phenotype and guarantee high efficiency of cell survival with minimal cell cross-contamination.

<table><tbody><tr><td>0.45 &amp;mu;m filter</td><td>Thermo Scientific</td><td>7211345</td><td>Preparing plate with collagen coating</td></tr><tr><td>10 cm cell culture plate</td><td>Greiner Bio-One</td><td>664160</td><td>Cell culture/cell line expansion</td></tr><tr><td>10 mL serological pipet</td><td>Fisher</td><td>14955234</td><td>VEC/VIC isolation, cell culture, cell line expansion</td></tr><tr><td>1000 &amp;mu;L filter tips</td><td>VWR</td><td>76322-154</td><td>Cell culture/cell line expansion</td></tr><tr><td>10XL filter tips</td><td>VWR</td><td>76322-132</td><td>Cell culture/cell line expansion</td></tr><tr><td>15 mL conical tubes</td><td>Thermo Scientific</td><td>339650</td><td>Tissue storage, VIC/VEC isolation</td></tr><tr><td>16% paraformaldehyde aqueous solution</td><td>Electron Microscopy Sciences</td><td>15710S</td><td>Tissue and cell fixative</td></tr><tr><td>190 proof ethanol</td><td>Decon</td><td>2801</td><td>Disinfection</td></tr><tr><td>1x DPBS: no calcium, no magnesium</td><td>Gibco</td><td>14190250</td><td>Saline solution. VIC/VEC isolation</td></tr><tr><td>1x PBS</td><td>Fisher</td><td>BP2944100</td><td>Saline solution. Tissue preparation, VIC/VEC isolation</td></tr><tr><td>20 &amp;mu;L filter tips</td><td>VWR</td><td>76322-134</td><td>Cell culture/cell line expansion</td></tr><tr><td>200 proof ethanol</td><td>Decon</td><td>2701</td><td>Deparaffinizing tissue samples</td></tr><tr><td>2-propanol</td><td>Fisher</td><td>A416P 4</td><td>Making collagen coated plates</td></tr><tr><td>5 mL serological pipet</td><td>Fisher</td><td>14955233</td><td>VEC/VIC isolation, cell culture, cell line expansion</td></tr><tr><td>50 mL conical tubes</td><td>Thermo Scientific</td><td>339652</td><td>Tissue storage, VIC/VEC isolation</td></tr><tr><td>60 mm dish</td><td>GenClone</td><td>25-260</td><td>VEC isolation</td></tr><tr><td>6-well cell culture plate</td><td>Corning</td><td>3516</td><td>Cell culture/cell line expansion</td></tr><tr><td>Acetic acid, glacial</td><td>Fisher</td><td>BP2401 500</td><td>Making collagen coated plates</td></tr><tr><td>AlexaFluor 488 phalloidin</td><td>Invitrogen</td><td>A12379</td><td>Fluorescent f-actin counterstain</td></tr><tr><td>Belzer UW Cold Storage Transplant Solution</td><td>Bridge to Life</td><td>BUW0011L</td><td>Tissue storage solution</td></tr><tr><td>Bovine Serum Albumin, Fraction V - Fatty Acid Free 25g</td><td>Bioworld</td><td>220700233</td><td>VEC confirmation with CD31+ Dynabeads</td></tr><tr><td>Calponin 1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab46794</td><td>Primary antibody (VIC positive stain)</td></tr><tr><td>CD31 (PECAM-1) (89C2)</td><td>Cell Signaling</td><td>3528</td><td>Primary antibody (VEC positive stain)</td></tr><tr><td>CD31+ Dynabeads</td><td>Invitrogen</td><td>11155D</td><td>VEC confirmation with CD31+ Dynabeads</td></tr><tr><td>CDH5</td><td>Cell Signaling</td><td>2500</td><td>Primary antibody (VEC positive stain)</td></tr><tr><td>Cell strainer with 0.70 &amp;mu;m pores</td><td>Corning</td><td>431751</td><td>VIC isolation</td></tr><tr><td>Collagen 1, rat tail protein</td><td>Gibco</td><td>A1048301</td><td>Making collagen coated plates</td></tr><tr><td>Collagenase II</td><td>Worthington Biochemical Corporation</td><td>LS004176</td><td>Tissue digestion. Tissue preparation, VIC/VEC isolation</td></tr><tr><td>Conflikt Ready-to-use Disinfectant Spray</td><td>Decon</td><td>4101</td><td>Disinfection</td></tr><tr><td>Countess II Automated Cell Counter</td><td>Invitrogen</td><td>A27977</td><td>Automated cell counter</td></tr><tr><td>Countess II reusable slide coverslips</td><td>Invitrogen</td><td>2026h</td><td>Automated cell counter required slide cover</td></tr><tr><td>Coverslips</td><td>Fisher</td><td>125485E</td><td>Mounting valve samples</td></tr><tr><td>Cryogenic vials</td><td>Olympus Plastics</td><td>24-202</td><td>Freezing cells/tissue samples</td></tr><tr><td>Disinfecting Bleach with CLOROMAX - Concentrated Formula&amp;nbsp;</td><td>Clorox</td><td>N/A</td><td>Disinfection</td></tr><tr><td>DMEM</td><td>Gibco</td><td>10569044</td><td>Growth media. VIC expansion</td></tr><tr><td>EBM - Endothelial Cell Medium, Basal Medium, Phenol Red free 500</td><td>Lonza Walkersville</td><td>CC3129</td><td>Growth media. VEC expansion</td></tr><tr><td>EGM-2 Endothelial Cell Medium-2 - 1 kit SingleQuot Kit</td><td>Lonza Walkersville</td><td>CC4176</td><td>Growth media supplement. VEC expansion</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>EVOS FL Microscope</td><td>Life Technologies</td><td>Model Number: AME3300</td><td>Fluorescent imaging</td></tr><tr><td>EVOS XL Microscope</td><td>Life Technologies</td><td>AMEX1000</td><td>Visualizing cells during cell line expansion</td></tr><tr><td>Fetal Bovine Serum - Premium Select</td><td>R&amp;amp;D Systems</td><td>S11550</td><td>VIC expansion</td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14088-10</td><td>Tissue preparation, VIC/VEC isolation</td></tr><tr><td>Fisherbrand Cell Scrapers</td><td>Fisher</td><td>08-100-241</td><td>VIC expansion</td></tr><tr><td>Fungizone</td><td>Gibco</td><td>15290-026</td><td>Antifungal: Tissue preparation, VIC/VEC isolation</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-064</td><td>Antibiotic: Tissue preparation, VIC/VEC isolation</td></tr><tr><td>Glass slides</td><td>Globe Scientific Inc</td><td>1358L</td><td>mounting valve samples</td></tr><tr><td>Goat anti-Mouse 488</td><td>Invitrogen</td><td>A11001</td><td>Fluorescent secondary Antibody</td></tr><tr><td>Goat anti-Mouse 594</td><td>Invitrogen</td><td>A11005</td><td>Fluorescent secondary Antibody</td></tr><tr><td>Goat anti-Rabbit 488</td><td>Invitrogen</td><td>A11008</td><td>Fluorescent secondary Antibody</td></tr><tr><td>Goat anti-Rabbit 594</td><td>Invitrogen</td><td>A11012</td><td>Fluorescent secondary Antibody</td></tr><tr><td>Invitrogen Countess II FL Reusable Slide</td><td>Invitrogen</td><td>A25750</td><td>Automated cell counter required slide</td></tr><tr><td>Invitrogen NucBlue Fixed Cell ReadyProbes Reagent (DAPI)</td><td>Invitrogen</td><td>R37606</td><td>Fluorescent nucleus counterstain</td></tr><tr><td>LM-HyCryo-STEM - 2X Cryopreservation media for stem cells</td><td>HyClone Laboratories, Inc.</td><td>SR30002</td><td>Frozen cell storage</td></tr><tr><td>Mounting Medium</td><td>Fisher Chemical Permount</td><td>SP15-100</td><td>Mounting valve samples</td></tr><tr><td>Mr. Frosty freezing container</td><td>Nalgene</td><td>51000001</td><td>Container for controlled sample freezing</td></tr><tr><td>Mycoplasma-ExS Spray</td><td>PromoCell</td><td>PK-CC91-5051</td><td>Disinfection</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140163</td><td>Antibiotic. VIC expansion</td></tr><tr><td>Plasmocin</td><td>Invivogen</td><td>ANTMPT</td><td>Anti-mycoplasma. VIC/VEC isolation and expansion</td></tr><tr><td>SM22a antibody</td><td>Abcam</td><td>ab14106</td><td>Primary antibody (VIC positive stain)</td></tr><tr><td>Sstandard pattern scissors</td><td>Fine Science Tools</td><td>14001-14</td><td>Tissue preparation, VIC/VEC isolation</td></tr><tr><td>Sterile cotton swab</td><td>Puritan</td><td>25806 10WC</td><td>VEC isolation</td></tr><tr><td>Swingsette human tissue cassette</td><td>Simport Scientific</td><td>M515-2</td><td>Tissue embedding container</td></tr><tr><td>Taylor Forceps (17cm)</td><td>Fine Science Tools</td><td>11016-17</td><td>Tissue preparation, VIC/VEC isolation</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>15250061</td><td>cell counting solution</td></tr><tr><td>TrypLE Express Enzyme</td><td>Gibco</td><td>12604021</td><td>Splitting VIC/VECs</td></tr><tr><td>Von Kossa kit</td><td>Polysciences</td><td>246331</td><td>Staining paraffin sections of tissues for calcification</td></tr><tr><td>von Willebrand factor antibody</td><td>Abcam</td><td>ab68545</td><td>Primary antibody (VEC positive stain)</td></tr><tr><td>Xylenes</td><td>Fisher Chemical</td><td>X3S-4</td><td>Deparaffinizing tissue samples</td></tr><tr><td>&amp;alpha;SMA antibody</td><td>Abcam</td><td>ab7817</td><td>Primary antibody (VIC positive stain)</td></tr></tbody></table>

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.

The above protocol outlines the steps necessary for the handling of human valve tissues and the isolation and establishment of viable cell lines from these tissues. Leaflets of the aortic valve are processed for paraffin embedding, snap frozen for long term storage for biochemical or genetic analysis and digested for the isolation of VECs and VICs (Figure 1). While surgical specimens will likely have a clinical diagnosis of aortic stenosis and may exhibit heavy nodules of calcification that ...

Watch this Scientific Journal Video about Isolation of Human Primary Valve Cells for In vitro Disease Modeling at JoVE.com

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

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-human-primary-valve-cells-for-in-vitro-disease-modeling

Research

JoVE Journal

Biology

2.7K Views.  University of Pittsburgh. De isolatie van menselijke primaire klep endotheel en interstitiële cellen is van cruciaal belang voor het begrijpen van het onderliggende mechanisme van de pathogenese van calcific aortaklepziekte. Wanneer de klep uit het oorspronkelijke weefsel wordt weggesneden, daalt de levensvatbaarheid van de cel. Dus hebben we een methode geïdentificeerd die de levensvatbaarheid van cellen verlengt.Na ontvangst van de geëxtraheerde klepweefselmonsters, extraheer de aortawortel en dompel het w...

Video: Isolation of Human Primary Valve Cells for In vitro Disease Modeling

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 ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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

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

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

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 ...

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 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.

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.

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

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

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/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. ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

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