This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

This protocol follows the LUMC guidelines of the animal research ethics committee.
1. Preparation of Instruments, Culture Medium, and MTCS
Note: Perform all preparations in the laminar flow hood. The MTCS perfusion chamber, bubble trap and stand are described in Lieber et al., 201011.
<ol>
	<li>Disinfect the forceps and micro scissors with 70% ethanol. Prepare a 5 ml syringe with 21G needle with sterile Tyrode&#8217;s Buffer (130 mM NaCl, 5.4 mM KCl, 6.0 mM Hepes, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM Glucose, 1.5 mM CaCl2.2H2O, pH=7.2). Prepare a 5 ml syringe with 21G needle with sterile KCl solution (100 mM).</li>
	<li>Prepare 65 ml of medium per heart: DMEM supplemented with 10% Fetal Calf Serum, antibiotic/antimycotic (A/A; 100 units of penicillin,100 &#956;g of streptomycin, and 0.25 ug of amphotericin B/ml), Insulin-Transferrin-Selenium (ITS; 10 &#956;g/ml insulin, 5.5 &#956;g/ml Transferrin, 6.7 ng/ml sodium selenite) and filter sterilize.</li>
	<li>Sterilize the perfusion chamber and bubble trap by spraying with 70% ethanol and dry with paper towel. Assemble the MTCS (Figure 1D), however, initially without the perfusion chamber. Position the bubble trap on the stand. Use a peristaltic roller pump with easy-load pump heads.
	<ol>
		<li>Using silicon tubing make connections between the reservoir (50 ml tube) and the pump, the pump and the bubble trap, the bubble trap and reservoir (see Figure 1D). Make the tubing inside the incubator long enough to allow medium to reach gas equilibrium with the gas in the incubator through the gas permeable silicon tubing (1 m) and the tubing outside the incubator as short as possible.</li>
	</ol>
	</li>
	<li>Fill the 50 ml tube with 70% ethanol, turn on the pump (flow speed around 1 ml/min to allow proper circulation) and let the ethanol circulate for 30 min&#160;to sterilize all tubing. Remove the ethanol by emptying the reservoir and pumping out the ethanol from the system.</li>
	<li>Fill the 50 ml tube with sterile distilled water, turn on the pump and let the water circulate for 30 min&#160;to get rid of remaining ethanol. Remove the water by emptying the reservoir and pumping out the water from the system.</li>
	<li>Fill the 50 ml tube with 45 ml of medium, put the stand in the incubator. Turn on the pump (flow speed around 1 ml/min) and circulate the medium to fill all tubing, remove all air bubbles and allow the medium to adapt to the gas composition in the incubator for at least 1 hr. Maintain the incubator at standard tissue-culture conditions (5% CO2 and 37 &#176;C). Ensure that no bubbles are present in the tubing.</li>
</ol>
2. Isolation of Mouse Heart
<ol>
	<li>Inject the mouse with 500 units of Heparin. This will prevent the blood from clotting and allows the removal of blood from the heart. After 10 min, anesthetize the mouse in an induction chamber with 4% isoflurane using a precision vaporizer. The anesthesia is sufficient when the mouse is unresponsive to toe pinch.</li>
	<li>Transfer the mouse to a dissection board and maintain anesthesia by means of a facemask connected to a coaxial circuit. Disinfect&#160;the fur of the mouse with&#160;70%&#160;alcohol.&#160;Using scissors open the abdominal cavity to expose the vena cava and diaphragm.</li>
	<li>To expose the heart, make lateral incisions starting from the last to the first ribs and reflect the thoracic cage over mouse&#8217;s head. Stop the anesthesia as mouse is not breathing anymore. Observe the heart beating.</li>
	<li>Insert the 21G needle attached to a 5 ml syringe containing sterile Tyrode&#8217;s Buffer into the inferior vena cava from the abdominal into the thoracic cavity (through diaphragm). Make sure that blood can exit from the vena cava caudal from the needle insertion. Perfuse the Tyrode&#8217;s Buffer gently and with constant pressure into the vena cava until the heart loses partly its red color. Note: The heart remains beating allowing the removal of the blood from the heart.</li>
	<li>Replace the needle with the 21G needle attached to a 5 ml syringe containing sterile KCl solution. Gently perfuse the KCl solution into the vena cava until the heart stops beating and remove the needle. Note: The KCl&#160;solution preserves the heart in the relaxed diastolic phase, which will allow easier insertion of the perfusion needles and perfusion of the coronary system.</li>
	<li>Slightly lift the heart using curved forceps and using scissors dissect the heart free from surrounding tissue but leave arteries and veins proximal to the heart intact and attached to the heart. Transfer the heart to a 15 ml tube containing ice-cold PBS supplemented with antibiotic/antimycotic (A/A). Store the heart in ice-cold PBS for up to 3 hr.</li>
</ol>
3. Cannulation of the Mouse Hearts in the Perfusion Chamber
<ol>
	<li>Perform all procedures in laminar flow hood. Transfer the isolated heart from the 15 ml tube to a 10 cm Petri dish and add PBS supplemented with A/A.</li>
	<li>Under a dissecting microscope, use the micro scissor and forceps to remove all non-cardiac tissue but preserve the ascending aorta to at least the bifurcation with the brachiocephalic artery and the pulmonary veins, 2&#160;mm proximal to the heart. Cut off the tip of the apex of the heart to create access to the left ventricular lumen (typically 2 mm). Place the perfusion chamber in the flow hood under the dissecting microscope.</li>
	<li>Attach a 5 ml syringe with medium to the insert needle using the silicone tubing. Fill the perfusion chamber with 20 ml medium and put the heart on the rotation stage in between the 2 blunted needles in the perfusion chamber (Figure 1). Adjust the height of the rotation stage so the heart is positioned in front of the needles.</li>
</ol>
4. Ligation for Culturing the Mitral Valve (see Figure 1)
<ol>
	<li>Ligate the aorta with suture (silk 7.0). Ligate the left atrial appendage with suture. Insert the needle (nr. 1 in Figure 1) through the pulmonary vein into the left atrium and ligate with suture proximal to its entry in the left atrium. Make sure the needle is not too far into the left atrium as this would damage the mitral valve.</li>
	<li>Insert the needle (nr.2 in Figure&#160;1) with linear motion into the left ventricle. Transfer the medium from the perfusion chamber to a 50 ml tube. Seal the needle to the myocardium with biocompatible glue.</li>
	<li>After the glue has dried, carefully inject medium into heart by connecting a medium-filled syringe to needle nr.1 and check if there is any leakage. Ensure that medium exits from needle nr.2 and the last remaining blood is perfused out of the heart.</li>
</ol>
5. Ligation for Culturing the Aortic Valve (see Figure 1)
<ol>
	<li>Insert the needle (nr.1 in Figure 1) in the aorta and ligate with suture. Make sure the needle is not too far into the aorta as this would damage the aortic valve. Insert the needle (nr.2 in Figure 1) with linear motion into the left ventricle. Transfer the medium from the perfusion chamber to a 50 ml tube. Seal the needle to the myocardium with biocompatible glue.</li>
	<li>After the glue has dried, carefully inject medium into the heart by connecting a medium-filled syringe to needle nr.2 and check if there is any leakage. Ensure that medium exits from needle nr.1 and the last remaining blood is perfused out of the heart.</li>
</ol>
6. Place Perfusion Chamber on Stand
<ol>
	<li>Fill up perfusion chamber with the 20 ml transferred medium. Place gasket and lid on the chamber and tighten with washer and screws.</li>
	<li>Remove the assembled MTCS from the incubator. Attach the perfusion chamber on the stand. Connect the tubing (see Figure 1D). Place the stand in the incubator (5% CO2 and 37 &#176;C). Connect the tubing to the pump. Turn on the pump with flow speed around 600 &#956;l/min.</li>
	<li>Make sure that in the reservoir and/or the bubble trap the level of the medium allows the visualizations of the presence of the flow by the falling of the incoming medium drops. After culture, the heart is removed from the system, fixed and processed for histological examination.</li>
</ol>

<ol>
	<li>Go, A. 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=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>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=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., 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=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

The authors declare no conflicts of interest.

Critical steps in culturing the cardiac mouse valves include making the time between excision of the heart from the mouse and the ligation in the perfusion chamber as short as possible to ensure viability and ligation of the needles perpendicular to the valves to ensure proper direction of the flow. Additionally, checking the flow after ligation in the perfusion chamber without medium ensures proper insertion and ligation of needles. It is critical to maintain a sterile culture and prevent air bubbles in the tubing, which could potentially get trapped in the heart obstructing the flow.
The total volume of medium used for the perfusion of a heart is 45 ml (note that the medium that is perfused through the heart is not in contact with the 20 ml of medium in the perfusion chamber). For the addition of for example viruses a smaller volume can be preferred. Temporally the reservoir (and part of the tubing) can be removed from the circuit leaving 5-7 ml in the system to allow infection of the valve.
The absence of beating of the heart could be considered a limitation. However, it allows creating an experimental condition in which all stimuli working on the leaflet can be kept constant. This gives the unique opportunity to study the effect of one single modification. When moving valves are required, a pulsatile flow can be created.
To study the valvular biology and the role of molecular, cellular and mechanical stimuli on the valve, modifications can be easily made to the culture system. Molecular modification can be achieved by the addition of growth factors, chemical compounds and viruses mediating gene delivery11 to the perfusion medium. One or multiple cell types can be administered to the perfusion medium to examine the influence of these cells on valvular biology or their contribution to the valve. The influence of oxidative stress or hypoxia can be studied by altering the oxygen-pressure in the culture medium. The hemodynamic stresses on the valve can be varied by changing the flow speed, flow direction, flow pulsatility and the viscosity. The overall morphology can be examined (Figure&#160;2B,C) by H&#38;E, histochemical and immunofluorescent staining. Additionally the organization and composition of the ECM, the phenotype, proliferation, and viability of the valvular cells and the activation of signaling pathways provide insight into the effects of varying these conditions. Genetically modified mice can be used to track the endothelial cells, using endothelial reporter mice (using the Tie2-Cre and floxed reporter mice) indicating the presence of endothelial-to-mesenchymal transformation or mice with loss or gain of function of specific signaling pathways. Overall, the system presented here is a useful tool for studying cardiac valvular biology.

Heart valve disease is a major cause of morbidity and mortality in the Western world; its prevalence increases with age and it affects more than 10% of the population 75 years and older1. The valves of the systemic part of the heart, the aortic and mitral valves, are mostly affected. Heart valve disease is characterized by the loss of the highly organized structure of the valves, which results in the alteration of the mechanical properties2. The structural integrity is therefore critical for the function of the valve.
The leaflets of the valve are comprised of valvular interstitial cells (VIC), valvular endothelial cells (VEC), and extracellular matrix, which is highly organized in an layered pattern3,4. The VICs are responsible for the ECM synthesis, degradation and organization. Factors emanating from the bloodstream, ECs or residing in the ECM act on the VICs orchestrating its function. In addition, mechanical forces act on the leaflet during the cardiac cycle resulting in laminar or oscillatory shear stress, compressive or tensile stresses influencing the behavior of VICs5.
In order to understand how the structure of the valve is regulated, it must first be understood how VICs respond to the diverse set of stimuli experienced during the cardiac cycle. In vitro studies have been very informative about the characteristics and abilities of the valvular cells. The response of these cells in vitro, however, may not always accurately mimic the in vivo response6; for example, the response of VIC to stimuli is dependent on the presence of ECs and the ECM composition5. Furthermore, the response of the valvular cells to stimuli depends on their specific location in the leaflet7. In addition to biochemical stimuli, the behavior of the valvular cells is determined by mechanical forces acting on the valve8. Each region of the valve is subjected to its own specific set of hemodynamic stresses. Although current ex vivo models have shown that mechanical forces are important determinants of valvular structure5, the associated mechanisms are still unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development9,10, insights into adult valvular biology is still elusive.
Therefore, an ex vivo flow model was developed in which the cardiac valves can be cultured in their natural position in the heart for an extended period of time11. This has the advantage that the valves remain in their natural configuration and the VICs experience the same environment as in vivo, making the VICs responses to stimuli as natural as possible. In addition, the culture of the valves in their natural position in the heart facilitates subjecting each valvular region to the relevant hemodynamic stresses. In this ex vivo model, i.e., the Miniature Tissue Culture System (MTCS), the valves can be subjected to different biochemical and hemodynamic stimuli allowing the investigation of their role in cardiac valve remodeling.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400-045</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Life Technologies</td><td>15240-06</td><td></td></tr><tr><td>Silk 7-0</td><td>Ethicon</td><td>768G</td><td></td></tr><tr><td>100 mm culture dish</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>50 ml tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>5 ml syringe</td><td>BD</td><td>309649</td><td></td></tr><tr><td>21 G needle</td><td>BD</td><td>304432</td><td></td></tr><tr><td>Heparin</td><td>LEO</td><td>012866-08</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-00</td><td></td></tr><tr><td>Micro Scissors, Economy, Vannas-type</td><td>Tedpella</td><td>1346</td><td></td></tr><tr><td>Silicon tubing</td><td>Thermo Scientific</td><td>8060-0020</td><td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td></tr><tr><td>Silicon tubing for pump</td><td>Masterflex</td><td>96400-13</td><td>I.D. x O.D. x Wall: 0.8 x1.59 x 0.40 mm</td></tr><tr><td>Biocompatible glue (Histoacryl)</td><td>B. Braun</td><td>1050071</td><td></td></tr><tr><td>precision vaporizer</td><td>Dr&amp;auml;ger</td><td>Vapor 200</td><td></td></tr><tr><td>peristaltic roller pump</td><td>Masterflex</td><td>7521-35</td><td></td></tr><tr><td>Easy-load pump head</td><td>Masterflex</td><td>7518-00</td><td></td></tr><tr><td>Flow chamber</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr><tr><td>Bubble trap</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr></tbody></table>

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.

The aortic valve (Figure 2) or mitral valve11 can be cultured for at least 3 days. By culturing in the open position (which represents the systolic position for the aortic valve and the diastolic position for the mitral valve), valvular cells remain viable. No cell death is observed as determined by the absence of TUNEL-positive cells (Figure 2H,I) or cleaved caspase-3 expression (not shown and Lieber et al., 201011). The collagen distribution (as visualized by Masson&#8217;s Trichrome staining) is similar to the native condition when cultured in 1% serum (Figure 2D,E). The valves are responsive to changing culture conditions. Increasing the amount of serum to 10% results in thicker leaflets (Figure 2C). Furthermore, a clear collagen-free region is observed at the ventricular side of the leaflet near the attachment to the aortic wall (arrow in Figure 2F). Together these observations show that the cultured valves remain viable, resemble their native counterparts and are responsive to environmental changes.
<img alt="Figure 1" src="/files/ftp_upload/52750/52750fig1.jpg" /> 
Figure 1. The Miniature Tissue Culture system. (A) In the perfusion chamber the heart is laying on the rotation stage and ligated with the blunted needles indicated with nr.1 and nr.2. (B) Perfusion chamber is mounted on the stand. (C) Schematic drawing of the heart when culturing the mitral valve (left) or aortic valve (right). (D) Schematic drawing of the setup of the ex vivo flow system for aortic valve culture. This figure has been partly modified from previous study11. <a href="https://www.jove.com/files/ftp_upload/52750/52750fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52750/52750fig2.jpg" /> 
Figure 2. Histological analysis of cultured valves from 2 months old mice. (A-C) heamatoxylin and eosin (H&#38;E) (D-F) Masson&#8217;s Trichrome staining and G-I) TUNEL staining of a non-cultured aortic valve (A,D,G), an aortic valve cultured in the presence of 1% serum (B,E,H) or 10% serum (C,F,I) for 3 days in the MTCS. The valve cultured with 1% serum appears similar to the non-cultured valve, whereas the valve cultured with 10% serum is thicker (C) and has an altered ECM pattern (F). No apoptosis is observed in the valves (G-I). Scale bar is 100 um. <a href="https://www.jove.com/files/ftp_upload/52750/52750fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

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 ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K Views.  Leiden University Medical Center. 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.

Video: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Medicine

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

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, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

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) ...

Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

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

Test zwapnienia do pomiaru stężenia wapnia w komórkach śródmiąższowych zastawki aortalnej

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

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Podsumowanie

Przeglądaj więcej filmów

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

In Vitro Model of Coronary Angiogenesis

Isolation of Murine Valve Endothelial Cells

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

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

Test zwapnienia do pomiaru stężenia wapnia w komórkach śródmiąższowych zastawki aortalnej