The authors would like to thank the Commission for Technology and Innovation (CTI, Switzerland) for their financial support (No. 13236.1 PFFLI-LS).

The investigations were performed using a piped pump setup (Figure&#160;2), which enables mechanical stress experiments at flow rates up to 60&#160;L&#160;min-1 and pressure drops up to 2&#160;bar to be performed. As shown in Figure&#160;2, the experimental setup is comprised of the storage vessel, the pump circuit, and the equipment for the inline endoscopy technique. The impeller of the storage vessel was only used to mix the surfactant. Peripheral elements were integrated into the closed loop to monitor the flow rate V&#775; and pressure drop p at different pump settings. The investigations were varied by using the hand-wheel valve.
1. Experimental Setup
<ol>
	<li>Ensure that the bioreactor (D&#160;=&#160;0.15&#160;m, H/D&#160;=&#160;2.2) is equipped with an impeller for the dissolution of the surfactant and make sure that the inlet tube dips into the fluid to avoid a gas entering.</li>
	<li>Equip the pump loop with a syringe port, the investigated pump, a clamp-on flow meter, a single-use pressure sensor and a hand-wheel valve. After connection of the pump loop to the storage vessel, connect the pump head to the motor and prepare the endoscope probe.</li>
	<li>Mount the changeable reflection plane, a rhodium mirror in this case, at the probe tip and adjust the distance between mirror and lens to 150&#160;&#181;m. Adjust the screw at 100&#160;&#181;m to focus the sharpness of the objective.</li>
	<li>Connect the probe to the stroboscope via a fiber-optic cable and the camera of the endoscope to the computer via an Ethernet cable. Then, connect the camera and the stroboscope together via a trigger-box cable.</li>
	<li>Start the computer and open the manufacturer-provided software, which includes an image acquisition and recognition software, as well as result analyzer software.</li>
	<li>Select the image acquisition software in the main menu.</li>
	<li>Click on the button &#8220;Detect device&#8221; in the upper left corner of the screen to detect the camera.</li>
	<li>Under &#8220;Directory Settings&#8221; select the location on the computer for saving images and activate the command &#8220;Create trigger sub folders&#8221;.</li>
	<li>Enter the process parameters in section &#8220;Trigger mode: Ready&#8221;. 
	Frame rate: 7.5&#160;Hz 
	Frames per trigger: 50 
	Number of triggers: 60 
	Trigger interval: 60&#160;sec</li>
	<li>After completion of all preparatory work, pour 5&#160;L deionized water into the storage vessel and switch on the pump to fill the pump and the pump loop.</li>
	<li>Switch off the pump and add 0.9 ml of surfactant (csurfactant&#160;=&#160;0.18&#160;ml L-1, &#961;surfactant,20&#176;C &#160;=&#160;1,070&#160;kg&#160;m-3, critical micelle concentration (cmc): &#969;cmc&#160;&#8776;&#160;0.018&#160;ml L-1, &#969;surfactant&#160;&#8776;&#160;10&#160;&#183;&#160;&#969;cmc) with a 10 ml pipette under stirring. After 10&#160;min the surfactant is completely dissolved.</li>
	<li>Switch off the impeller and turn on the pump. Position the endoscope probe so that the lens is located directly beneath the inlet tube.</li>
	<li>Set the flow rate of 3.4&#160;L&#160;min-1 and the pressure drop of 0.03, 0.3 or 0.61&#160;bar by varying the impeller speed and the hand-wheel valve.</li>
	<li>Weigh 6.3&#160;g of oil directly in the syringe (&#946;oil&#160;=&#160;1.26&#160;g&#160;L-1, &#961;oil,20&#176;C&#160;=&#160;989.5&#160;kg&#160;m-3).</li>
	<li>Start the image acquisition software and add the oil via the syringe port. The running pump distributes the emulsion drops.</li>
	<li>After 1 hr, finish the shear stress investigation and clean the inline endoscope as well as the bioreactor with the integrated pump loop. Subsequently, prepare the experimental setup for the next pumping process.</li>
</ol>
2. Measurement and Image Analysis
<ol>
	<li>Open the automatic image recognition software in the main menu.</li>
	<li>Under &#8220;Batch Root Directory&#8221; select the location on the computer for saving the files (all*.csv).</li>
	<li>Select the column &#8220;Image Series Path&#8221; and click on the button &#8220;Add Image Series Subfolders&#8221; in the lower left corner of the screen to load the image series.</li>
	<li>Load the process parameters which are provided by the manufacturer. Select the column &#8220;Search Settings (*.pss or auftrag_*.mat)&#8221; and click on the button &#8220;Set Search Settings&#8221; in the lower-middle of the screen to load process parameters in order to specify the drop recognition.</li>
	<li>Select the column &#8220;Search Pattern (*.psp or F_*.mat) and click on the button &#8220;Set Search Pattern&#8221; in the lower right corner of the screen to load the process parameters in order to specify the drop analysis.</li>
	<li>Start the image recognition by clicking on the button &#8220;Start Batch&#8221;.</li>
	<li>After completion of image recognition, express the detected drop sizes by the Sauter mean diameter (d32), or any other representative mean value or distribution of choice by using the result analyzer software.</li>
	<li>Open the result analyzer software in the main menu.</li>
	<li>Activate the command &#8220;all*.csv in 1 Folder&#8221; and click on the button &#8220;Load Folder(s)&#8221; in the upper left corner of the screen to load the previously saved all*.csv file.</li>
	<li>Select the relevant value (e.g., Sauter mean diameter) in the drop-down list in the upper middle of the screen to visualize the results. For the calculation of the diameter enter the scaling of 0.6591&#160;&#181;m pixel-1 on the right, which is provided by the manufacturer.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53052/53052fig2.jpg" /> 
Figure 2. Experimental setup. Pump circuit for piped pump setup using inline endoscopy as the measurement technique: (1) storage vessel, (2) syringe port, (3) pump, (4) pressure sensor, (5) flow sensor, (6) stroboscope, (7) computer with the manufacturer-provided software, and (8) endoscope probe. <a href="https://www.jove.com/files/ftp_upload/53052/53052fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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=Response+of+a+concentrated+monoclonal+antibody+formulation+to+high+shear.">Response of a concentrated monoclonal antibody formulation to high shear.</a> Biotechnol. 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Wolfgang Dornfeld and Reto Sch&#246;b are employees of Levitronix, Ltd., manufacturer of the MagLev centrifugal pumps.

The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. For this purpose, drop sizes of an oil-water emulsion system were determined and a measured Sauter mean diameter was calculated to characterize the mechanical stress of the MagLev centrifugal pumps as well as their counterparts, a peristaltic and a 4-piston diaphragm pump. The results showed low variation of the measured Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating that drop sizes have been reliably and accurately measured. As a consequence, the measured Sauter mean diameter could successfully be used as a comparison criterion to evaluate the mechanical stress of the pumps investigated. The MagLev centrifugal pumps revealed larger measured Sauter mean diameters, indicating lower mechanical stresses on emulsion drops compared with the peristaltic and 4-piston diaphragm pumps. In studies to date, inline endoscopy has been shown to be a robust and simple technique for reliable drop size measurement1,6,12-14,20-21, which was also confirmed by this study. In comparison to alternative measurement techniques, such as the fiber optical FBR sensor, the FBRM and the 2D-ORM technique, the endoscope technique can be used as the standard method for obtaining precise data in liquid/liquid applications12,14.
The easy handling of the inline endoscopy and the simple production of the non-biological oil-water emulsion system enables a straightforward procedure for drop size detections according to the protocol text (see above). Nevertheless, it should be mentioned that the position of the endoscope probe depends on the fluid flow in the storage vessel. Further investigations (data not shown) have revealed that the lens of the probe should be located directly beneath the inlet tube for lower flow rates up to 5&#160;L&#160;min-1 in order to avoid a multiple detection of one drop19. For sharp images at flow rates over 5&#160;L&#160;min-1, it is recommended to position the probe at least 10&#160;cm away from the inlet tube. Independent of process parameters, the holder of the inline endoscopy should be stable in order to avoid a shifting of the probe, which can result in blurred images.
Furthermore, it should particularly be noted that the drop size detected is close to the lower detection limit of the applied photo-optical system, where the minimum detectable drop diameter is 6.5&#160;&#181;m. As the manufacturer-provided software has been improved, inline endoscopy techniques can reliably detect a minimal drop size of 1&#160;&#181;m. Moreover, the image processing will be further developed to enable online monitoring of industrial applications.
While the present study focused on relatively low flow rates of up to 3.4&#160;L&#160;min-1, future studies should consider a wider range of operation conditions. First investigations have been carried out at flow rates up to 20&#160;L&#160;min-1 (data not shown). However, a 1:2 dilution (csurfactant&#160;=&#160;0.09&#160;ml L-1, coil&#160;=&#160;0.64&#160;ml L-1) of the oil-water emulsion system is recommended at flow rates over 10&#160;L&#160;min-1&#160;19, as increased drop breakup caused by higher mechanical stress would otherwise affect drop detection and reduce the number of drops detected. Tests were carried out with a 1:2 dilution and compared with results of an undiluted oil-water emulsion system. For both approaches, the Sauter mean diameters have been reliably measured (standard deviation below 5%). Therefore, reduced volume fraction (1:2 dilution) did not influence the measured Sauter mean diameters, and thus a drop-drop breakup was negligible.
These powerful experimental approaches provide a good basis for the improvement of the endoscopy technique as well as the related image acquisition, recognition and result analyzer software. Furthermore, the suitability of the endoscopy technique to classify pump types and series according to their mechanical stress was successfully demonstrated. The results obtained are essential for pump design development and the optimization of pumps to reduce cell damage.

Pumps are used to transfer cell cultures in the pharmaceutical and biotechnological industries. During the pumping process, mechanical stress can result in irreversible cell damage, which might impair the quantity and quality of the product1-4. The level of mechanical stress depends on the pump type and pump settings, as demonstrated in previous studies5-6. Commonly, peristaltic, syringe and diaphragm pumps are used for single-use (SU) technology based applications. These pumps result in high local shear forces caused by the compression of the pump tubing and the pulsating flow7.
In order to overcome these drawbacks, magnetically levitated centrifugal pumps (MagLev centrifugal pumps) constitute a promising alternative. The motor is magnetically driven in order to avoid narrow gaps between the impeller and the pump housing (Figure 1). A previous study investigated the MagLev centrifugal pumps and showed lower mechanical stress in Chinese Hamster Ovary (CHO) cells compared with peristaltic and 4-piston diaphragm pumps5. In addition, hemolysis analyses revealed no significant blood trauma and thrombus formation over a range of operation conditions using these pumps8-11. The findings demonstrate that use of these specifically designed pumps applies less mechanical stress on biological systems in comparison with peristaltic and diaphragm pumps. To investigate the mechanical stress with limited experimental expense, an oil-water emulsion model system is recommended due to its cost- (ca. 99.8%) and time-reduced (ca. 99.5%) application compared with biological cell culture systems.
As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes must be counted over the experimental time of shear stress investigations. Many techniques for sizing drops are available, which can be divided into sound, laser and photo based techniques12. In particular, the use of the photo-optical probe inline endoscopy shows almost identical drop sizes for manual and automatic detections (standard deviation below 10%) and enables a detection of 250 drops per minute13. Because of its accuracy and reliability, the endoscope technique has been shown to be an effective standard measurement technique for drop size distributions in liquid/liquid dispersions when compared with other commonly used probes (e.g., fiber optical forward-backward-ratio (FBR) sensor, focused beam reflectance method (FBRM) and the two-dimensional optical reflectance measurement technique (2D-ORM))12,14. Moreover, the suitability of inline endoscopy for measuring drop sizes in a stirred vessel has been demonstrated several times in previous investigations15-18.
Based on a prior study6, this protocol describes the use of inline endoscopy to determine drop sizes (Sauter mean diameter) of an oil-water emulsion system in pumps. The Sauter mean diameter was used as a comparison criterion in order to estimate the mechanical stress of the multi-use (MU) MagLev centrifugal pumps, a peristaltic and a single-use (SU) 4-piston diaphragm pump.
<img alt="Figure 1" src="/files/ftp_upload/53052/53052fig1.jpg" /> 
Figure 1. Magnetically levitated centrifugal pump-system. (A) The principle of a bearingless motor and (B) the PuraLev&#160;200MU are shown as an example. <a href="https://www.jove.com/files/ftp_upload/53052/53052fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>CCD camera</td><td>Allied Vision Technologies GmbH</td><td>GX2750</td><td>Equipment for inline endoscopy</td></tr><tr><td>C-Flex Biopharmaceutical Tubing</td><td>Saint-Gobain Performance Plastics</td><td>374-375-4</td><td>Tube&lt;br/&gt; Select a tubing length of about 45&amp;nbsp;cm before the pump.</td></tr><tr><td>C-Flex Biopharmaceutical Tubing</td><td>Saint-Gobain Performance Plastics</td><td>374-375-3</td><td>Tube&lt;br/&gt; Select a tubing length of about 45&amp;nbsp;cm after the pump and clamp on the flow sensor to this tubing.</td></tr><tr><td>CLAVE Connector</td><td>Victus</td><td>011-C2000</td><td>Sampling port</td></tr><tr><td>Controller LPC-200.1-02</td><td>Levitronix GmbH</td><td>100-30030</td><td>PuraLev 200MU controller</td></tr><tr><td>Controller LPC-600.1-02</td><td>Levitronix GmbH</td><td>100-30033</td><td>PuraLev 600MU controller</td></tr><tr><td>LeviFlow Clamp-On Sensor LFSC-12</td><td>Levitronix GmbH</td><td>100-30329</td><td>Flow sensor for flow rates below 5&amp;nbsp;L&amp;nbsp;min&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>LeviFlow Converter LFC-1C-CS</td><td>Levitronix GmbH</td><td>100-30328</td><td>Flow sensor output device&amp;nbsp;</td></tr><tr><td>Masterflex I/P Easy Load</td><td>Fisher Scientific AG</td><td>EW-77963-10</td><td>Peristaltic pump</td></tr><tr><td>Mitos free flow valve</td><td>Parker Hannifin Europe S&amp;agrave;rl</td><td>FFLQR16S6S6AM</td><td>Valve</td></tr><tr><td>Mobil Eal Arctic</td><td>Exxon Mobil Corporation</td><td>Mobil EAL Arctic 22</td><td>Oil&lt;br/&gt; Prepare the emulsion directly before the experiment.</td></tr><tr><td>Motor</td><td>Elektromotorenwerk Brienz AG</td><td>7WAC72N4THTF</td><td>Motor for agitator shaft</td></tr><tr><td>Motor BSM-1.4</td><td>Levitronix GmbH</td><td>100-10005</td><td>PuraLev 200MU motor</td></tr><tr><td>Motor LPM-600.4</td><td>Levitronix GmbH</td><td>100-10038</td><td>PuraLev 600MU motor</td></tr><tr><td>Norm-Ject 10 ml Luer Lock</td><td>Restek Corporation</td><td>22775</td><td>Syringe</td></tr><tr><td>Pump Head LPP-200.5</td><td>Levitronix GmbH</td><td>100-90525</td><td>PuraLev 200MU pump head</td></tr><tr><td>Pump Head LPP-600.18</td><td>Levitronix GmbH</td><td>100-90548</td><td>PuraLev 600MU pump head</td></tr><tr><td>Quattroflow 1200-SU</td><td>Almatechnik AG</td><td>QF 1200</td><td>4-piston diaphragm pump</td></tr><tr><td>SciPres Sensor</td><td>SciLog</td><td>080-695PSX</td><td>Pressure sensor</td></tr><tr><td>SciPres Sensor Monitor</td><td>SciLog</td><td>080-690</td><td>Pressure sensor output device&amp;nbsp;</td></tr><tr><td>SOPAT-VF Inline Endoscopic Probe</td><td>SOPAT GmbH</td><td></td><td>Inline endoscopy</td></tr><tr><td>Stroboscope</td><td>Drello GmbH &amp;amp; Co KG</td><td>Drelloscop 255-01</td><td>Equipment for inline endoscopy</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Surfactant&lt;br/&gt;Handle with gloves and goggles.&lt;br/&gt; (acute toxicity, eye irritation)</td></tr></tbody></table>

a cost-effective and reliable method to predict mechanical stress in single-use and standard pumps

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping process shear forces occur which can lead to qualitative and/or quantitative product loss. To calculate the mechanical stress with limited experimental expense, an oil-water emulsion system was used, whose suitability was demonstrated for drop size detections in bioreactors1. As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes need to be counted over the experimental time of shear stress investigations. In previous studies, the inline endoscopy has been shown to be an accurate and reliable measurement technique for drop size detections in liquid/liquid dispersions. The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. In order to express the drop size, the Sauter mean diameter d32 was used as the representative diameter of drops in the oil-water emulsion. The results showed low variation in the Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating the reliability of the measurement technique.

Optical assessment
Figure&#160;3 shows the particle recognition images after a pumping time of 1 hr. The upper four images show the drops before recognition and the lower four images show the drops marked by the recognition software. The drops detected are highlighted with a green edge. Comparing the upper and lower images shows that the drop edges were precisely detected by the image recognition software. The images on the left show the drop distribution for the MagLev centrifugal pumps PuraLev&#160;200MU and PuraLev&#160;600MU, and those on the right show the 4-piston diaphragm and the peristaltic pump. An optical assessment allowed an initial classification of the mechanical stress in the model emulsion system. This revealed that larger drop sizes and lower drop counts were generated by the MagLev centrifugal pumps compared with the 4-piston diaphragm and peristaltic pump. Consequently, the MagLev centrifugal pumps, especially the PuraLev&#160;200MU, showed reduced drop breakage, indicating lower mechanical stresses.
<img alt="Figure 3" src="/files/ftp_upload/53052/53052fig3.jpg" /> 
Figure 3. Images of the inline endoscopy. Emulsion drops before (A, B, C, D) and after (E, F, G, H) particle recognition after 1 hr of pumping using (A, E) the PuraLev&#160;200MU, (B, F) the PuraLev&#160;600MU, (C, G) the 4-piston diaphragm pump, and (D, H) the peristaltic pump under identical operating conditions (3.4&#160;L&#160;min-1 and 0.03&#160;bar). <a href="https://www.jove.com/files/ftp_upload/53052/53052fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sauter mean diameter
Further investigations revealed standard deviations below d32&#160;&#177;&#160;0.4&#160;&#181;m and guaranteed reproducible results when using inline endoscopy19. Therefore, multiple investigations were not required for this approach, which additionally reduced the experimental expense.
In order to express the drop size, the Sauter mean diameter d32 (see Eq.&#160;1) was used as the representative diameter of drops in the oil-water emulsion for this approach. In general, the Sauter mean diameter decreased over time for all pump types and pump settings until reaching steady state12. Investigations in this study confirmed the progression of the Sauter mean diameter (Figure&#160;4A to D), curves of the PuraLev&#160;200MU (Figure&#160;4A) and the peristaltic pump (Figure&#160;4D) being exemplarily discussed in this protocol.
In contrast to the PuraLev&#160;200MU, the Sauter mean diameters were up to 40% smaller for the peristaltic pump at the same operation conditions (flow rate&#160;=&#160;3.4&#160;L&#160;min-1; pressure drop&#160;=&#160;0.03&#160;bar). As a consequence, higher mechanical stresses resulted in an increased drop breakup and therefore smaller drop sizes. Furthermore, the Sauter mean diameter decreased with increasing pressure drop in the PuraLev&#160;200MU (Figure&#160;4A), which indicated the dependency of drop size on pressure drop. In contrast, the peristaltic pump showed a Sauter mean diameter of d32,60min&#160;=&#160;10&#160;&#181;m at the end of the experiment for all process parameters (Figure&#160;4D). Therefore, the Sauter mean diameter was found to be independent of pressure drop. However, the results reflect physical understanding of the drop breakup: with higher mechanical stress, smaller Sauter mean diameters were determined (see also Figure&#160;5).
For each measurement point, at least 300&#160;drops were determined in order to guarantee statistical certainty. The maximal standard deviation decreased for the PuraLev&#160;200MU from d32,4min&#160;&#177;&#160;42&#160;&#181;m and for the PuraLev&#160;600MU from d32,6min&#160;&#177;&#160;21&#160;&#181;m to approximately d32&#160;&#177;&#160;0.5&#160;&#181;m at the end of the pumping process. The decreased standard deviation resulted from the increased homogeneous drop size distribution until steady state was reached. In comparison to the MagLev centrifugal pumps, the peristaltic and 4-piston diaphragm pumps revealed standard deviations below d32&#160;&#177;&#160;10&#160;&#181;m.
<img alt="Equation 1" src="/files/ftp_upload/53052/53052eq1.jpg" width="80" /> (1)
<img alt="Figure 4" src="/files/ftp_upload/53052/53052fig4.jpg" /> 
Figure 4. Typical profiles of Sauter mean diameters d32 over time and the determination of the measured Sauter mean diameters d32,m. Comparison of Sauter mean diameters d32 (A) for the PuraLev&#160;200MU, (B) for the PuraLev&#160;600MU, (C) for the 4-piston diaphragm pump, and (D) for the peristaltic pump. The Sauter mean diameters d32 were determined at a flow rate of 3.4&#160;L&#160;min-1 and pressure drops ranging from 0.03 to 0.61&#160;bar. The measured Sauter mean diameter d32,m was calculated for the last 10 min&#160;(boundary). The resulting standard deviation of the Sauter mean diameters d32 (N&#160;&#8805;&#160;300) is shown. <a href="https://www.jove.com/files/ftp_upload/53052/53052fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Measured Sauter mean diameter as a comparison system
As described above, the Sauter mean diameter decreased over time until the drop sizes reached steady state. In the last 10&#160;min of the experimental time, the average value of the Sauter mean diameter was calculated to determine the measured Sauter mean diameter, which was used as a comparison criterion (see boundary of Figure&#160;4A-D). The measured Sauter mean diameters d32,m are shown for a flow rate of 3.4&#160;L&#160;min-1 and a pressure drop range from 0.03 to 0.61&#160;bar in Figure&#160;5. Larger measured Sauter mean diameters were determined for both MagLev centrifugal pumps (200MU and 600MU) and the 4-piston diaphragm pump at lower pressure drops and impeller speeds. The peristaltic pump revealed measured Sauter mean diameters of d32,m&#160;=&#160;10&#160;&#181;m for all process parameters. As previously mentioned, shear forces were independent of the pressure drop for the peristaltic pump.
The largest measured Sauter mean diameters of d32,m&#160;=&#160;36&#160;&#181;m for the PuraLev&#160;200MU and d32,m&#160;=&#160;34&#160;&#181;m for the PuraLev&#160;600MU were obtained at a pressure drop of 0.03&#160;bar. In comparison with their counterparts, the MagLev centrifugal pump series obtained up to 59% larger measured Sauter mean diameters. These results indicated a lower rate of drop breakup and thus lower mechanical stress resulting from the use of the centrifugal pumps.
The standard deviation of the measured Sauter mean diameters during the steady state was below 15%, thereby confirming reliable and accurate values for the drop sizes.
<img alt="Figure 5" src="/files/ftp_upload/53052/53052fig5.jpg" /> 
Figure 5. Comparison of measured Sauter mean diameters d32,m. Measured Sauter mean diameters for the MagLev centrifugal pumps and their counterparts at 3.4&#160;L&#160;min-1 and pressure drops of 0.03, 0.30 and 0.61&#160;bar. The resulting standard deviations of the measured Sauter mean diameters d32,m during the steady state are shown.
Abbreviations <a href="https://www.jove.com/files/ftp_upload/53052/53052fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2D-ORM</td>
			<td>two-dimensional optical reflectance measurement</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>charge-coupled device</td>
		</tr>
		<tr>
			<td>CHO</td>
			<td>Chinese hamster ovary</td>
		</tr>
		<tr>
			<td>cmc</td>
			<td>critical micelle concentration</td>
		</tr>
		<tr>
			<td>FBR</td>
			<td>forward-backward-ratio</td>
		</tr>
		<tr>
			<td>FBRM</td>
			<td>focused beam reflectance method</td>
		</tr>
		<tr>
			<td>MU</td>
			<td>multi-use</td>
		</tr>
		<tr>
			<td>SU</td>
			<td>single-use</td>
		</tr>
	</tbody>
</table>
Nomenclature
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td><img alt="V" fo:content-="" src="/files/ftp_upload/53052/V.jpg" width="8
" /></td>
			<td>[m3 sec-1]</td>
			<td>flow rate</td>
		</tr>
		<tr>
			<td>c</td>
			<td>[m3 m-3]</td>
			<td>concentration</td>
		</tr>
		<tr>
			<td>d32</td>
			<td>[m]</td>
			<td>Sauter mean diameter</td>
		</tr>
		<tr>
			<td>d32,m</td>
			<td>[m]</td>
			<td>measured Sauter mean diameter</td>
		</tr>
		<tr>
			<td>ds</td>
			<td>[m]</td>
			<td>surface diameter</td>
		</tr>
		<tr>
			<td>dv</td>
			<td>[m]</td>
			<td>volume diameter</td>
		</tr>
		<tr>
			<td>f</td>
			<td>[Hz]</td>
			<td>frequency</td>
		</tr>
		<tr>
			<td>n</td>
			<td>[sec-1]</td>
			<td>impeller speed</td>
		</tr>
		<tr>
			<td>N</td>
			<td>[-]</td>
			<td>number of drops</td>
		</tr>
		<tr>
			<td>p</td>
			<td>Pa</td>
			<td>pressure drop</td>
		</tr>
		<tr>
			<td>t</td>
			<td>sec</td>
			<td>time</td>
		</tr>
		<tr>
			<td>&#946;</td>
			<td>[kg m-3]</td>
			<td>mass concentration</td>
		</tr>
		<tr>
			<td>&#961;</td>
			<td>[kg m-3]</td>
			<td>density</td>
		</tr>
		<tr>
			<td>&#969;</td>
			<td>[m3 m-3]</td>
			<td>mass fraction</td>
		</tr>
	</tbody>
</table>
Table 1. Table of abbreviations and nomenclature.

Watch this Scientific Journal Video about A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps at JoVE.com

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol.

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping ...

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9.4K Views.  Zurich University of Applied Sciences. Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol. 

Video: A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Bioengineering

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Modeling and Experimental Analysis of the Single-Shaft Coaxial Motor-Pump Assembly in Electrohydrostatic Actuators

An electrohydrostatic actuator (EHA) can be the most promising alternative compared with the traditional hydraulic servo actuators for its high power density, ease of maintenance, and reliability. As the core power unit that determines the performance and service life of the EHA, the motor-pump assembly should simultaneously possess a wide speed/pressure range and a high dynamic response.
This paper presents a method to test the performance of the motor-pump assembly through simulation and experimentation. The flow output characteristics were defined through simulation and analysis of the assembly at the beginning of the experiment, leading to the conclusion of whether the pump could meet the requirements of the EHA. A series of performance tests were conducted on the motor-pump assembly via a pump test bench in the speed range of 1,450-9,000 rpm and the pressure range of 1-30 MPa.
We tested the overall efficiency of the motor-pump assembly under various working conditions after confirming the consistency between the test results of the flow output characteristics with the simulation results. The results showed that the assembly has higher overall efficiency when working at 4,500-7,000 rpm under the pressure of 10-25 MPa and at 2,000-2,500 rpm under 5-15 MPa. Overall, this method can be utilized for determining in advance whether the motor-pump assembly meets the requirements of EHA. Moreover, this paper proposes a rapid test method of the motor-pump assembly in various working conditions, which could assist in predicting EHA performance.

We built a simulation model to evaluate pump flow characteristics and performance of the single-shaft coaxial motor-pump assembly in electrohydrostatic actuators and investigate the overall efficiency in a wide set of working conditions of the motor-pump assembly experimentally.

An electrohydrostatic actuator (EHA) can be the most promising alternative compared with the traditional hydraulic servo actuators for its high power ...

A Modeling and Simulation Method for Preliminary Design of an Electro-Variable Displacement Pump

Electro-hydrostatic actuators (EHAs) have been considerably researched in academia, and their applications in various industrial fields are expanding. The variable-speed EHA has now taken priority over the variable-displacement EHA, but its driving motor and associated electronics encounter issues when applied in high-power applications: low-dynamics, high thermal dissipation, high price, etc. Therefore, a variable-displacement EHA equipped with an electro-variable displacement pump (EVDP) has been considered. The EVDP itself is a mechatronic system that integrates a piston pump, a ball screw, a gearbox, and a permanent magnet synchronous motor (PMSM). Consequently, the EVDP needs to be investigated to ensure its system-level performance when applied in an EHA. In addition to the previous research on the technical parameters of the EVDP, a dedicated design method is necessary for further reducing the cost of using the EVDP and exploring its performance potential. Here, a simulation based EVDP preliminary design method is selected for designing a 37 kW EVDP. Firstly, a previously proposed multidisciplinary model of the EVDP is extended by improving the parameter generation, including the EVDP life, reliability, control models, etc. Secondly, the proposed model is partially verified using a downsized prototype. Thirdly, the EVDP is simulated at a system level, supported by the proposed model. The EVDP performance is evaluated according to the specified design requirements. The temperature, bandwidth and accuracy, reliability and lifetime, etc., are all predicted for the EVDP. The simulation results demonstrate the EVDP's applicability in variable-displacement EHA. The proposed modeling and simulation method can be used to evaluate diverse EVDP performance and respond to general design requirements. The method can also support the resolution of the preliminary design challenges in terms of limited information and robustness. Therefore, the proposed method is appropriate for the realization of the simulation-based EVDP preliminary design method.

A simulation model specifically supporting the preliminary design of an electro-variable displacement pump (EVDP) is developed and partially verified by experiments. The control performance, life, reliability, etc., can all be evaluated using the proposed model, which covers the main performance requirements under the EVDP preliminary design task.

Electro-hydrostatic actuators (EHAs) have been considerably researched in academia, and their applications in various industrial fields are expanding. The ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Immunology and Infection

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

Summary

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In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

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