The authors are grateful to Shad Schipke and Daniel Chesser for their assistance with the design and fabrication of the system and Valtresa Myles for assistance with preparing the manuscript.

1.	Tissue Harvest and Preparation
<ol>
<li>Aortic valves should be collected from adult pigs weighing no more than 120 lbs immediately after death.</li>
<li>Wash the valves twice with sterile phosphate buffered saline (PBS) and transport to the laboratory on ice.</li>
<li>All subsequent steps should be performed under sterile conditions.</li>
<li>Ensure that leaflets do not show any sign of degeneration, tearing or calcification. Remove leaflets from the aortic root by cutting 1/3 of the distance from the annulus.</li>
<li>Place leaflets in individual wells of a six well plate and incubate overnight with 3ml Dulbecco's Modified Eagle Medium supplemented with 1% anti-biotic/anti-mycotic solution and 10% fetal bovine serum at 37&deg;C and 5% CO2.</li>
<li>As an alternative, isolated cells can be seeded into six-well culture plates and used in the pressure device. Isolation of valve endothelial cells and interstitial cells can be performed as previously described13, 14.</li>
</ol>
2.	Pressure Studies
<ol>
<li>A custom-made pressure system has been designed to study the mechanobiological effects of cyclic pressure on aortic valve tissue15.</li>
<li>Log on to the computer and open the labVIEW program (Figure 1).</li>
<li>Calibration:<ol>
<li>Prior to experimentation, the system should be properly calibrated.</li>
<li>Connect the power supply to the circuit board. This provides power to the solenoid valves that control the flow of air into and out of the chamber.</li>
<li>Make sure the compressed air is connected to the system and open the air supply to full velocity.</li>
<li>Turn on the signal amplifier. Make sure the voltage reading is 0.00. Adjust as necessary</li>
<li>The labVIEW interface has a switch marked "TEST/RECORD". Make sure the switch is set to "TEST". Click the button marked "Air Supply" to open the inlet solenoid valve.</li>
<li>Using the gas pressure regulator, pressurize the chamber with compressed air at 1 PSI. The pressure in the chamber can be read using the digital pressure gauge located on the rear end-plate of the chamber. Once the pressure has equilibrated, record the voltage reading from the signal amplifier. Repeat for 2, 3, 4 and 5 PSI.</li>
<li>Construct a calibration curve of pressure vs. voltage. Pressure should be converted from PSI to mmHg. The equation from the graph can be placed into the code of the labVIEW program</li>
</ol>
</li>
<li>Remove the aluminum front plate from the pressure chamber and spray the chamber with 70% ethanol. Leave for a minimum of 10 minutes to allow residual ethanol fumes to dissipate.</li>
<li>Place the six-well plate containing the leaflet samples into the chamber and replace the front end plate. Ensure the seal is airtight by tightening the nuts located on the four threaded rods (located at each corner of the end plate) by hand. Place the pressure chamber in the 37&deg;C incubator. A schematic diagram of the pressure chamber is shown in figure 2.</li>
<li>The interface will prompt the user to provide the amount of time the system cycles between compressed air input and output. These should be set to 0.6s and 0.4s to mimic diastolic and systolic conditions, respectively, at a frequency of 1Hz. The user should also enter a data file path.</li>
<li>In labVIEW, click run and switch the "TEST/RECORD" toggle to "RECORD".</li>
<li>Make sure the pressure is at the desired level using the graph on the labVIEW interface. Pressure can be adjusted using the gas pressure regulator.</li>
<li>Run program for desired length of time.</li>
<li>Once the experiment is complete, click the stop button on labVIEW, turn off air supply and open the exhaust valve on the pressure chamber.</li>
<li>Remove the front end plate from the chamber and retrieve six-well plate containing samples. Samples can now be analyzed for gene expression, protein expression, histology, mechanical properties etc.</li>
</ol>
3.	Representative Results: 
The pressure system is capable of simulating the maximum transvalvular pressures observed under normotensive, stage I and stage II hypertensive conditions. However, the pressure was not able to mimic the systolic pressure gradient, which is essentially zero in vivo. Frequency is maintained at 1Hz, with an air inlet time of 0.6s and an exhaust time of 0.4s. Representative pressure waveforms of normal and elevated pressure conditions obtained from the system can be seen in figure 3. 
<img src="/files/ftp_upload/3316/3316fig1.jpg" alt="Figure 1"/> Figure 1: Screen shot of the LabVIEW interface.
<img src="/files/ftp_upload/3316/3316fig2.jpg" alt="Figure 2"/> Figure 2: Schematic drawing of the pressure chamber A. Isometric view of pressure chamber; B. Side view of pressure chamber; C. Top view of pressure chamber.
<img src="/files/ftp_upload/3316/3316fig3.jpg" alt="Figure 3"/> Figure 3: Graph of pressure simulation within the pressure chamber at (A) normotensive, (B) Stage I hypertensive, and (C) Stage II hypertensive conditions.

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No conflicts of interest declared.

The pressure system successfully exposed aortic valve leaflets to cyclic pressures that were representative of diastolic transvalvular pressure. However, it was not able to mimic systolic transvalvular pressure, as the pressure only dropped to 40 mmHg. Transvalvular pressure is the difference between pressure in the ascending aorta and the left ventricle. During diastole, when the valve is closed, the pressure difference is 80mmHg under normotensive conditions and 90 mmHg and 100mmHg in stage I and stage II hypertension,...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's PBS</td>
			<td>Sigma</td>
			<td>D5652</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mycotic/antibiotic solution</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoScientific</td>
			<td>SH30070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Viton diaphragm solenoid valves</td>
			<td>McMaster Carr</td>
			<td>4868K11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pressure Transducer		</td>
			<td>Omega Engineering, Inc.</td>
			<td>PX302-200GV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Load cell conditioner		</td>
			<td>Encore Electronics, Inc.</td>
			<td>4025-101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Data Acquisition (DAQ) Module		</td>
			<td>Measurement Computing</td>
			<td>PMD1608</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

design of a cyclic pressure bioreactor for the ex vivo study of aortic heart valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Watch this Scientific Journal Video about Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves at JoVE.com

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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15.6K Views.  Mississippi State University. A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.