Name: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System (Video) | JoVE Journal
Uploaded: 2015-10-19T14:00:00.000+00:00
Duration: 8 min 47 s
Description: Learn to culture mouse cardiac valves using the miniature tissue culture system (MTCS) for cardiac biology research. This method enables histological analysis of valve viability, morphology, and extracellular matrix distribution.

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5 minutes

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/pubmed?term=((Heart+Disease+and+Stroke+Statistics--2014+Update%3A+A+Report+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">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/pubmed?term=((Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling%5BTitle%5D)+AND+%22Developmental+biology%22%5BJournal%5D)">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/pubmed?term=((Cardiac+valves+and+valvular+pathology%3A+update+on+function%2C+disease%2C+repair%2C+and+replacement%5BTitle%5D)+AND+%22Cardiovascular+pathology%3A+the+official+journal+of+the+Society+for+Cardiovascular+Pathology%22%5BJournal%5D)">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/pubmed?term=((Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification%5BTitle%5D)+AND+%22International+journal+of+inflammation%22%5BJournal%5D)">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/pubmed?term=((Mechanobiology+of+the+Aortic+Heart+Valve%5BTitle%5D)+AND+%22The+Journal+of+heart+valve+disease%22%5BJournal%5D)">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/pubmed?term=((An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch%5BTitle%5D)+AND+%22Annals+of+biomedical+engineering%22%5BJournal%5D)">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/pubmed?term=((Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets%5BTitle%5D)+AND+%22Journal+of+Biomechanics%22%5BJournal%5D)">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/pubmed?term=((Heart+valve+development%3A+Regulatory+networks+in+development+and+disease%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">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/pubmed?term=((TGF%CE%B2+and+BMP+signaling+in+cardiac+cushion+formation%3A+lessons+from+mice+and+chicken%5BTitle%5D)+AND+%22Differentiation%3B+research+in+biological+diversity%22%5BJournal%5D)">TGFβ 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/pubmed?term=((Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves%5BTitle%5D)+AND+%22Annals+of+biomedical+engineering%22%5BJournal%5D)">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, whic...

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

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 visualize...

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 ...

The overall goal of this procedure is to culture mouse cardiac valves using the miniature tissue culture system or MTCS. This is accomplished by first assembling the MTCS without the profusion chamber and circulating the medium to precondition the system. In the second step, the mouse heart is harvested and prepared for cannulation.Next, the heart is placed in the profusion chamber where it is cannulated for a culture of the mitral or aortic valve. The perfusion chamber is then placed in the MTCS and the pump has started. Ultimately, histological analysis is used to assess the viability, morphology and extracellular matrix distribution of the valve.Begin by using pieces of silicone tubing to make connections between the reservoir and the pump. The pump and the bubble trap, and the bubble trap and the reservoir. When all of the tubing is connected, fill the reservoir with 70%ethanol.Turn the pump on at one milliliter per minute, flow speed, and let the ethanol circulate to sterilize the tubing. After 30 minutes, empty the reservoir and pump the ethanol out of the system. Next, fill the reservoir with sterile distilled water and pump the water through the system to remove the residual alcohol.Then pump the water out again and fill the reservoir with 45 milliliters of freshly prepared medium. Circulate the medium to fill all of the tubing and allow the medium to adapt to the gas composition in the incubator for at least one hour. Under standard tissue culture conditions.To isolate the mouse heart, first, inject the animal with 500 units of heparin ip. After 10 minutes, place the mouse on a dissection board and confirm the appropriate level of anesthesia by toe. Pinch, sterilize the fur with 70%ethanol, and then use a pair of scissors to open the abdominal cavity, exposing the venina cava and diaphragm to visualize the heart, make lateral incisions starting from the bottom and ending at the top of the ribs.Then reflect the thoracic cage over the animal's head and observe the heart beating. Next, insert a 21 gauge needle attached to a five milliliter syringe containing sterile tyros buffer through the diaphragm into the inferior vena cva from the abdominal into the thoracic cavity. Make sure that the blood can exit from the vena cva, coddle from the needle insertion, and then perfused the buffer gently and with constant pressure into the vena cava until the heart partly loses its red color.Now switch out the syringe of buffer for five milliliters of sterile potassium chloride solution, and gently perfuse the solution into the vena CVA until the heart stops beating. Using curved forceps and scissors, slightly lift and dissect the heart from the surrounding tissue. Then keep the organ in a 10 milliliter tube containing ice cold PBS supplemented with antibiotics and anti mycotic for up to three hours to cannulate the heart, transfer it into a 10 centimeter Petri dish within a laminar flow hood, and add fresh PBS supplemented with antibiotics and antimycotic Using a dissecting microscope, micro scissors and forceps, remove all of the non-cardiac tissue, preserving the ascending aorta to at least the bifurcation with the brachiocephalic artery and the pulmonary veins, two millimeters proximal to the heart.Then cut two millimeters off of the tip of the apex to create access to the left ventricular lumen and placed a perfusion chamber in the hood using pieces of silicone tubing. Next, attach five milliliter syringes filled with medium to the number one needles for culture of the mitral valve, and number two, needles for culture of the aortic valve. And fill the profusion chamber with 20 milliliters of medium.Then put the heart on the rotation stage in the perfusion chamber between the two blunted needles, and adjust the height of the stage so that the heart is positioned in front of the needles to culture. The mitral valve ligate the aorta and the left atrial appendage with a silk 7.0 suture. Then insert the needle number one through the pulmonary vein into the left atrium, and place a suture proximal to its entry in the left atrium.Taking care that the needle does not enter too far into the left atrium, insert the needle number two into the left ventricle. Then transfer the medium from the profusion chamber into a 50 milliliter tube, and use biocompatible glue to seal the needle to the myocardium. When the glue is dry, use the needle number one syringe to carefully inject the medium into the heart.Check the heart for leakage and confirm that the medium exits from the needle. Number two, and that the remaining blood is perfused out of the tissue. To culture the aortic valve, insert the needle number one into the aorta and ligate it with a suture as just demonstrated taking care not to place the needle too far into the aorta.Then insert the needle number two, into the left ventricle, seal the needle and inject the medium as just demonstrated. If there is no leakage, fill the profusion chamber with 20 milliliters of the transferred medium. Place the gasket and the lid on the chamber and tighten both with a washer and screws.Place the profusion chamber onto the stand. Finally, place the profusion chamber into the stand and start the pump. With a flow speed of around 600 microliters per minute, the aortic and mitral valves can be cod for at least three days with no cell death observed during the culture as determined by the absence of tunnel positive cells.Further, the collagen distribution as visualized by Massen Trichome staining is similar to that observed for the native condition. When cultured in 1%serum, the valves are responsive to changing culture conditions. For example, as seen here, increasing the amount of serum to 10%results in thicker leaflets.Furthermore, a clear collagen free region is observed at the ventricular side of the leaflet near the attachment to the aortic wall. Together these observations demonstrate that the cultured valves remain viable, resembled their native counterparts, and are responsive to environmental changes after its development. This technique paved the way for researchers in the field of valvular biology to explore the mechanisms that regulate valvular homeostasis and disease.

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Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

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