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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+aortic+valve+stenosis:+TGF-beta1+is+present+in+calcified+aortic+valve+cusps+and+promotes+aortic+valve+interstitial+cell+calcification+via+apoptosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+cells+in+calcified+aortic+valves+have+reduced+differentiation+potential+and+stem+cell-like+properties.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aortic+valve+interstitial+cells+display+proangiogenic+properties+during+calcific+aortic+valve+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+transcriptional+regulatory+mechanisms+in+aortic+valve+development+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+of+aortic+valve+stenosis+induced+by+direct+wire+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+calcific+aortic+valve+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+valvular+endothelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+valve+endothelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hyperinsulinemia+and+hyperglycemia+on+valvular+interstitial+cells+-+A+link+between+aortic+heart+valve+degeneration+and+type+2+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+trends+in+the+incidence+and+prognosis+of+aortic+stenosis:+A+nationwide+study+of+the+Swedish+population.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+and+reversible+changes+of+interstitial+cell+phenotype+during+remodeling+of+cardiac+valves.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+induction+of+adenosine+receptor+genes+and+its+functional+significance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+transcriptome+atlas+of+human+aortic+valves+reveal+cell+heterogeneity+and+endothelial+to+mesenchymal+transition+involved+in+calcific+aortic+valve+disease.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.45 &#956;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 &#956;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 &#956;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&#160;</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 &#956;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&#160;</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 rowspan="2" >Lonza Walkersville</td>
			<td rowspan="2">CC4176</td>
			<td rowspan="2" >Growth media supplement. VEC expansion</td>
		</tr>
		<tr >
			<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&#38;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 >&#945;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

2.5K Views.  University of Pittsburgh. 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. 

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

Isolation and Primary Culture of Rat Hepatic Cells

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

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

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

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

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 and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

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) ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...